Therapeutic systems

ABSTRACT

A compound comprising a target cell-specific portion and human NAD(P)H:quinone reductase 2 (NQO2) or a variant or fragment or fusion or derivative thereof which has substantially the same activity as NQO2 towards a given prodrug, or a polynucleotide encoding said NQO2 or said variant or fragment or fusion or derivative. A recombinant polynucleotide comprising a target cell-specific promoter operably linked to a polynucleotide encoding human NAD(P)H:quinone reductase 2 (NQO2) or a variant or fragment or fusion or derivative thereof which has substantially the same activity as NQO2 towards a given prodrug. The compounds and polynucleotides are useful in a method of treating a patient in conjunction with a suitable prodrug. A method of treating a human patient with a target cell to be destroyed wherein the target cell expresses NQO2 the method comprising administering to the patient a prodrug which is converted to a substantially cytotoxic drug by the action of NQO2 and nicotinamide riboside (reduced) (NRH) or an analogue thereof which can pass reducing equivalents to NQO2.

Priority is claimed under 35 U.S.C. §119 to PCT/GB98/01731, filed Jun.15, 1998, which claims priority to GB9712370.7 filed Jun. 14, 1997.

The present invention relates to therapeutic systems, particularlytherapeutic systems for activating prodrugs and for the use of suchsystems in king target cells, particularly tumour cells.

The delivery of a cytotoxic agent to the site of tumour cells is muchdesired because systemic administration of these agents can result inthe killing of normal cells within the body as well as the tumour cells.The resulting toxicity to normal cells limits the dose of the cytotoxicagent and thus reduces the therapeutic potential of these agents.However, in some instances the administered agent has no intrinsicactivity but is converted in vivo at the appropriate time or place tothe active drug. Such analogues are referred to as prodrugs and are usedextensively in medicine [Connors and Knox, 1995]. Conversion of theprodrug to the active form can take place by a number of mechanismsdepending, for example, on changes of pH, oxygen tension, temperature orsalt concentration or by spontaneous decomposition of the drug orinternal ring opening or cyclisation.

WO 88/07378 describes a two-component system, and therapeutic usesthereof, wherein a first component comprises an antibody fragmentcapable of binding with a tumour-associated antigen and an enzymecapable of converting a pro-drug into a cytotoxic drug, and a secondcomponent which is a prorug which is capable of conversion to acytotoxic drug. This general system, which is often referred to as“antibody-directed enzyme pro-drug therapy” (ADEPT), is also describedin relation to specific enzymes and pro-drugs in EP 0 302 473 and WO91/11201.

WO 89/10140 describes a modification to the system described in WO88107378 wherein a further component is employed in the system. Thisfurther component accelerates the clearance of the first component fromthe blood when the first and second components are administeredclinically. The second component is usually an antibody that binds tothe antibody-enzyme conjugate and accelerates clearance. An antibodywhich was directed at die active site on the enzyme had the additionaladvantage of inactivating the enzyme. However, such an inactivatingantibody has the undesirable potential to inactivate enzyme at thetumour sites, but its penetration into turnouts was obviated by theaddition of galactose residues to the antibody. The galactosylatedantibody was rapidly removed from the blood, together with boundantibody-enzyme component, via galactose receptors in the liver. Thesystem has been used safely and effectively in clinical trials. However,galactosylation of such an inactivating antibody which results in itsrapid clearance from blood also inhibits its penetration of normaltissue and inactivation of enzyme localised there.

WO 93/13805 describes a system comprising a compound comprising a targetcell-specific portion, such as an antibody specific to tumour cellantigens, and an inactivating portion, such as an enzyme, capable ofconverting a substance which in its native state is able to inhibit theeffect of a cytotoxic agent into a substance which has less effectagainst said cytotoxic agent. The prolonged action of a cytotoxic agentat tumour sites is therefore possible whilst protecting normal tissuesfrom the effects of the cytotoxic agent. WO 93/13806 describes a furthermodification of the ADEPT system comprising a three component kit ofparts for use in a method of destroying target cells in a host. Thefirst component comprises a target cell-specific portion and anenzymatically active portion capable of converting a pro-drug into acytotoxic drug; the second component is a pro-drug convertible by saidenzymatically active portion to the cytotoxic drug; and the thirdcomponent comprises a portion capable of at least partly restraining thecomponent from leaving the vascular compartment of a host when saidcomponent is administered to the vascular compartment, and aninactivating portion capable of converting the cytotoxic drug into aless toxic substance.

Our unpublished but co-pending patent application GB 9624993.3 describesa macromolecule prodrug therapy system. Our unpublished but co-pendingpatent application PCT/GB96/03000 describes the use of enzyme inhibitorsin an improvement of ADEPT; and our unpublished but co-pending patentapplication PCT/GB96/03254 describes the use of internalising antibodiesand/or intracellular cofactors in an improvement to ADEPT.

EP 0 415 731 describes a therapeutic system which is often called GDEPT(gene-directed enzyme prodrug therapy).

A major approach in prodrug design is the synthesis of inert analogueswhich are converted to the active drug by enzyme action. In cancerchemotherapy prodrugs have been used clinically for a variety ofpurposes ranging from analogues with better formulation properties toprodrugs designed to be selectively activated in the tumour environment.Results from animal experiments and dose intensification studies inhumans have indicated that some tumour types, eg ovarian cancer, mightbe completely eradicated by chemotherapy if the dose of anti-canceragent to which they respond could be increased by a hundred-fold.Attempts to increase the dose administered using dose intensification,by autologous bone marrow transplantation after high doses of myelotoxictherapy, by rescue experiments eg folinic acid after methotrexate or byisolated limb perfusions, do allow a greater total dose to be given butnot by this order of magnitude. However, there are many examples wherethis level of dose intensity can theoretically be achieved by usingprodrugs which are selectively activated by enzymes present in tumours.Experiments on tumour bearing animals have shown that when a prodrug isactivated uniquely in the tumour environment, cures can be obtained formice bearing large primary tumours and extensive metastases [Connors andWhisson, 1966, Whisson and Connors, 1965]. Given that the prodrug is agood substrate for the enzyme specifically expressed in the tumour andthat the difference in toxicity between prodrug and drug is ahundred-fold or more then, once a candidate enzyme has been identified(especially if there is also a high concentration of the enzyme in theextracellular spaces of the tumour), many different classes ofanti-cancer agent can often be derivatised to form appropriate prodrugs.This can be demonstrated in approaches used to design prodrugs ofcytotoxic alkylating agents. Because this class of anti-cancer agentacts predominantly but probably not exclusively by covalent alkylationof adjacent strands of DNA the first basic requirement for cytotoxicityis that the agent should have an optimal level of chemical reactivitywhich enables it to reach the tumour site after injection and bereactive enough to alkylate DNA. If the reagent is too reactive it mayhydrolyse before reaching the tumour and if too unreactive may beexcreted before sufficient DNA alkylation has taken place. Secondly, itmust be able to pass through the endothelium and the cell and nuclearmembranes to reach its target. Finally, because the predominant reactionthat leads to cytotoxicity is a cross-linking reaction, then thealkylating agent must have a minimum of two alkylating arms.

In order to design an appropriate prodrug, once a unique tumour enzymehas been identified, a prodrug is synthesised which is lacking one ormore of the features described but is acted upon by the enzyme toproduce an appropriate drug. Thus, many alkylating prodrugs arechemically unreactive and non-toxic but are substrates for enzymes whichmetabolise them to highly reactive and toxic products. The ability of analkylating agent to react with biological molecules depends on a minimallevel of chemical reactivity and this level of activity can vary greatlydepending on chemical structure. Small changes in electron donating orwithdrawing properties can greatly alter chemical reactivity. Largenumbers of anti-tumour alkylating agents have been tested experimentallyand, almost without exception, active derivatives must be at leastdifunctional, ie have at least two alkylating arms.

Monofunctional agents, although they may be carcinogens, usually aremuch less toxic and if they can be converted enzymatically todifunctional agents might be effective prodrugs. An example of this isCB 1954 a monofunctional aziridine which was highly effective againstthe rat Walker tumour which is normally only sensitive to difunctionalalkylating agents (reviewed by Knox et al, 1993). This tumour has arelatively high concentration of the enzyme DT-diaphorase (NQO1, EC1.6.99.2) which reduces the 4-nitro group to a hydroxylamine which isthen converted (probably by acetyl CoA) to a difunctional agent (FIG.1). However, the human form of DT-diaphorase reduces CB 1954 much moreslowly than the rat form and human tumours (even those containing thesame levels of DT-diaphorase as rat Walker tumour) are resistant to thisagent [Knox et al, 1993]. The difference in reduction rate is mostly dueto a single amino-acid change, a glutamine to a tyrosine at amino acidposition 104 [Chen et al, 1997]. Given the provenance of CB 1954 againstrat tumours, a number of ways of activating CB 1954 in human tumourshave been suggested. The first is antibody directed enzyme prodrugtherapy (ADEPT, as mentioned above) in which an antibody is used tolocalise an E. coli nitroreductase at a tumour. This nitroreductase canreduce CB 1954 much more rapidly than rat DT-diaphorase. The system isdescribed in WO 93/08228. Gene directed enzyme prodrug therapy (GDEPT)is a method by which the gene encoding for the nitroreductase fromEscherichia coli is expressed in tumour cells and thus conferssensitivity to CB 1954. This is described in WO 95/12678.

It has also been reported that CB 1954 cytotoxicity can be dramaticallyincreased in human cells by stimulating their endogenous DT-diaphorasewith NRH [Friedlos et al, 1992a]. In these experiments, the toxicity ofCB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) towards human cells wasgreatly enhanced by NADH (when foetal calf serum was present in theculture medium) and by nicotinamide riboside (reduced) (NRH), but not bynicotinate riboside (reduced). Co-treatment of human cells with CB 1954and NADH resulted in the formation of crosslinks in their DNA. Thetoxicity produced by other DNA crosslinking agents was unaffected byreduced nicotinamide compounds. When caffeine was included in the mediuma reduction of the cytotoxicity of CB 1954 occurred. The toxicityexperienced by human cell lines after exposure to CB 1954 and NADH wasproportional to their levels of the enzyme DT diaphorase. It wasconcluded that NRH, which has been shown to be a cofactor for rat DTdiaphorase [Friedlos et al, 1992b], is generated from NADH by enzymes infoetal calf serum [Friedlos and Knox, 1992] and stimulates the activityof human DT diaphorase towards CB 1954. However, it has recently beenshown that there is an additional CB1954-reducing activity detectable inhuman cells in the presence of NRH and that this activity is muchgreater than that attributable to DT-diaphorase [Quinn, 1996].

The terms “nicotinamide mononucleoside-reduced”, “dihydronicotinamideriboside”, “nicotinamide riboside (reduced)” and “NRH” are allequivalent and are used interchangeably in the patent specification.Nicotinamide riboside may be produced enzymatically from itscommercially available mononucleotides using methods well known in theart, including those described in Friedlos & Knox (1992) Biochem.Plzarmacol. 44, 631-635 which is incorporated herein by reference.

Human NAD(P)H:quinone oxidoreductase2 (NQO2) was identified by itshomology to DT-diaphorase (NQO1) [jaiswal et al, 1990]. The last exon inthe NQO2 gene is 1603 bp shorter than the last exon of the NQO1 gene andencodes for 58 amino acids as compared to 101 amino acids encoded by theNQO1 gene. This makes the NQO2 protein 43 amino acids shorter than theNQO1 protein. The high degree of conservation between NQO2 and NQO1 geneorganization and sequence confirmed that the NQO2 gene encoded for asecond member of the NQO gene family in humans but it lacked thequinone-reductase activity characteristic of DT-diaphorase [Jaiswal,1994]. The NQO2 cDNA-derived protein expressed in monkey kidney COS1cells efficiently catalyzed nitroreduction of CB 10-200, an analogue ofCB 1954 [Jaiswal, 1994]. Northern blot analysis indicated that the NQO2gene was expressed in human heart, brain, lung, liver, and skeletalmuscle but did not express in placenta. In contrast, the NQO1 gene wasexpressed in all human tissues. Large variations were noticed forexpression of the NQO2 and NQO1 genes among various tissues [Jaiswal,1994].

We have now shown that NQO2 can rapidly reduce CB1954 and consider thisenzyme, not DT diaphorase, to be responsible for the potentiatingeffects of NRH on CB1954 cytotoxicity toward human cells reported byFriedlos et al [1992a].

Although all of the aforementioned methods of killing a target cell,such as a tumour cell, in an animal body are useful, it is stilldesirable to provide new systems of treatment.

A first aspect of the invention provides a compound comprising a targetcell-specific portion and (a) human NAD(P)H:quinone reductase 2 (NQO2)or a variant or fragment or fusion or derivative thereof which hassubstantially the same activity as NQO2 towards a given prodrug, or (b)a polynucleotide encoding said NQO2 or said variant or fragment orfusion or derivative.

The entity which is recognised by the target cell-specific portion maybe any suitable entity which is expressed by tumour cells,virally-infected cells, pathogenic microorganisms, cells introduced aspart of gene therapy or normal cells of the body which one wishes todestroy for a particular reason. The entity should preferably be presentor accessible to the targeting portion in significantly greaterconcentrations in or on cells which are to be destroyed than in anynormal tissues of the host that cannot be functionally replaced by othertherapeutic means. Use of a target expressed by a cancer cell would notbe precluded, for example, by its equal or greater expression on anendocrine tissue or organ. In a life-saving situation the organ could besacrificed provided its function was either not essential to life, forexample in the case of the testes, or could be supplied by hormonereplacement therapy. Such considerations would apply, for instance, tothe thyroid gland, parathyroids, adrenal cortex and ovaries.

The entity which is recognised will often be an antigen.Tumour-associated antigens, when they are expressed on the cell membraneor secreted into tumour extra-cellular fluid, lend themselves to therole of targets for antibodies.

The antigen-specific portion may be an entire antibody (usually, forconvenience and specificity, a monoclonal antibody), a part or partsthereof (for example an Fab fragment or F(ab′)₂) or a synthetic antibodyor part thereof. A conjugate comprising only part of an antibody may beadvantageous by virtue of optimizing the rate of clearance from theblood and may be less likely to undergo non-specific binding due to theFc part. Suitable monoclonal antibodies to selected antigens may beprepared by known techniques, for example those disclosed in “MonoclonalAntibodies: A manual of techniques”, H. Zola (CRC Press, 1988) and in“Monoclonal Hybridoma Antibodies: Techniques and Applications”, J. G. R.Hurrell (CRC Press, 1982). All references mentioned in thisspecification are incorporated herein by reference. Bispecificantibodies may be prepared by cell fusion, by reassociation ofmonovalent fragments or by chemical cross-linking of whole antibodies,with one part of the resulting bispecific antibody being directed to thecell-specific antigen and the other to the enzyme. The bispecificantibody can be administered bound to the enzyme or it can beadministered first, followed by the enzyme. It is preferred that thebispecific antibodies are administered first, and after localization tothe tumour cells, the enzyme is administered to be captured by thetumour localized antibody. Methods for preparing bispecific antibodiesare disclosed in Corvalan et al (1987) Cancer Immunol. Immunother. 24,127-132 and 133-137 and 138-143, and Gillsland et al (1988) Proc. Natl.Acad. Sci. USA 85, 7719-7723.

The variable heavy (V_(H)) and variable light (V_(L)) domains of theantibody are involved in antigen recognition, a fact first recognised byearly protease digestion experiments. Further confirmation was found by“humanisation” of rodent antibodies. Variable domains of rodent originmay be fused to constant domains of human origin such that the resultantantibody retains the antigenic specificity of the rodent parentedantibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81,6851-6855).

That antigenic specificity is conferred by variable domains and isindependent of the constant domains is known from experiments involvingthe bacterial expression of antibody fragments, all containing one ormore variable domains. These molecules include Fab-like molecules(Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al(1988) Science 240, 1038); single-chain Fv (ScFv) molecules where theV_(H) and V_(L) partner domains are linked via a flexible oligopeptide(Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl.Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprisingisolated V domains (Ward et al (1989) Nature 341, 544). A general reviewof the techniques involved in the synthesis of antibody fragments whichretain their specific binding sites is to be found in Winter & Milstein(1991) Nature 349, 293-299.

By “ScFv molecules” we mean molecules wherein the V_(H) and V_(L)partner domains are linked via a flexible oligopeptide.

The advantages of using antibody fragments, rather than wholeantibodies, are several-fold. The smaller size of the fragments may leadto improved pharmacological properties, such as better penetration ofsolid tissue. Effector functions of whole antibodies, such as complementbinding, are removed. Fab, Fv, ScFv and dAb antibody fragments can allbe expressed in and secreted from E. coli, thus allowing the facileproduction of large amounts of the said fragments.

Whole antibodies, and F(ab′)₂ fragments are “bivalent”. By “bivalent” wemean that the said antibodies and F(ab′)₂ fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv and dAb fragments aremonovalent, having only one antigen combining sites. Fragmentation ofintact immunoglobulins to produce F(ab′)₂ fragments is disclosed byHarwood et al (1985) Eur. J. Cancer Clin. Oncol. 21, 1515-1522.

IgG class antibodies are preferred.

Alternatively, the entity which is recognised may or may not beantigenic but can be recognised and selectively bound to in some otherway. For example, it may be a characteristic cell surface receptor suchas the receptor for melanocyte-stimulating hormone (MSH) which isexpressed in high numbers in melanoma cells. The cell-specific portionmay then be a compound or part thereof which specifically binds to theentity in a non-immune sense, for example as a substrate or analoguethereof for a cell-surface enzyme or as a messenger.

Considerable work has already been carried out on antibodies andfragments thereof to tumour-associated antigens and antibodies orantibody fragments directed at carcinoembryonic antigen (CEA) andantibodies or their fragments directed at human chorionic gonadotrophin(hCG) can be conjugated to carboxypeptidase G2 and the resultingconjugate retains both antigen binding and catalytic function. Followingintravenous injection of these conjugates they localise selectively intumours expressing CEA or hCG respectively. Other antibodies are knownto localise in tumours expressing the corresponding antigen.

Such tumours may be primary and metastatic colorectal cancer (CEA) andchoriocarcinoma (hCG) in human patients or other forms of cancer.Although such antibody-enzyne conjugates may also localise in somenormal tissues expressing the respective antigens, antigen expression ismore diffuse in normal tissues. Such antibody-enzyme conjugates may bebound to cell membranes via their respective antigens or trapped byantigen secreted into the interstitial space between cells.

Examples of tumour-associated, immune cell-associated and infectionreagent-related antigens are given in Table 1.

TABLE 1 Cell surface antigens for targeting Antigen Antibody Existinguses a) Tumour Associated Antigens Carcino-embryonic C46 (Amersham)Imaging and therapy Antigen 85A12 (Unipath) of colon/rectum tumours.Placental Alkaline H17E2 (ICRF, Imaging and therapy Phosphatase Travers& Bodmer) of testicular and ovarian cancers. Pan Carcinoma NR-LU-10(NeoRx Imaging and therapy Corporation) of various carci- nomasincluding small cell lung cancer. Polymorphic HMFG1 (Taylor- Imaging andtherapy Epithelial Mucin Papadimitriou, ICRF) of ovarian cancer and(Human milk fat pleural effusions. globule) β-human Chorionic W14Targeting of Gonadotropin carboxypeptidase to human xenograftchoriocarcinoma in nude mice (Searle et al (1981) Br. J. Cancer 44,137-144). A carbohydrate on L6 (IgG2a)¹ Targeting of alkaline HumanCarcinomas phosphatase (Senter et al (1988) PNAS USA 85, 4842-4846. CD20Antigen on B 1F5 (IgG2a)² Targeting of alkaline Lymphoma (normalphosphatase (Senter et and neoplastic) al (1988) PNAS USA 85, 4842-4846.¹Hellström et al (1986) Cancer Res. 46, 3917-3923 ²Clarke et al (1985)Proc. Natl. Acad. Sci. USA 82, 1766-1770

Other antigens include alphafoetoprotein, Ca-125 and prostate specificantigen

Antigen Antibody Existing uses b) Immune Cell Antigens Pan T LymphocyteQKT-3 (Ortho) As anti-rejection Surface Antigen therapy for kidney (CD3)transplants. B-lymphocyte RFB4 (Janossy, Immunotoxin therapy SurfaceAntigen Royal Free Hospital) of B cell lymphoma. (CD22) Pan T lymphocyteH65 (Bodmer and Immunotoxin Surface Antigen Knowles, ICRF; treatment ofacute (CD5) licensed to Xoma graft versus host Corp., USA) disease,rheumatoid arthritis. c) Infectious Agent-Related Antigens Mumps virus-Anti-mumps Antibody conjugated related polyclonal to diphtheria toxinantibody for treatment of mumps. Hepatitis B Surface Anti HBs AgImmunotoxin against Antigen hepatoma.

Other tumour selective targets and suitable binding moieties are shownin Table 2.

TABLE 2 Binding moieties for tumour-selective targets andtumour-associated antigens Target Binding moiety Disease Truncated EGFRanti-EGFR mAb Gliomas Idiotypes anti-id mAbs B-cell lymphomas EGFR(c-erbB1) EGF, TGFα anti- Breast cancer EGFR mAb c-erbB2 mAbs Breastcancer IL-2 receptor IL-2 Lymphomas and anti-Tac mAb leukaemias IL-4receptor IL-4 Lymphomas and leukaemias IL-6 receptor IL-6 Lymphomas andleukaemias MSH (melanocyte- α-MSH Melanomas stimulating hormone)receptor Transferrin receptor Transferrin anti-TR Gliomas (TR) mAbgp95/gp97 mAbs Melanomas p-glycoprotein cells mAbs drug-resistantcluster-1 antigen mAbs Small cell lung (N-CAM) carcinomas cluster-w4mAbs Small cell lung carcinomas cluster-5A mAbs Small cell lungcarcinomas cluster-6 (LeY) mAbs Small cell lung carcinomas PLAP(placental mAbs Some seminomas alkaline phosphatase) Some ovarian; somenon small cell lung cancer CA-125 mAbs Lung, ovarian ESA (epithelialmAbs carcinoma specific antigen) CD 19, 22, 37 mAbs B-cell lymphomas 250kDa mAbs Melanoma proteoglycan p55 mAbs Breast cancer TCR-IgH fusionmAbs Childhood T-cell leukaemia Blood gp A antigen mAbs Gastric andcolon (in B or O tumours individuals) Mucin protein core mAbs Breastcancer

It is preferred if the target cell-specific portion comprises anantibody or fragment or derivative thereof.

The target cell-specific portion may, however, be any compound whichleads to the accumulation of the NQO2 or a said variant or fragment orfusion or derivative thereof at the site of the target cell (such as atumour). For example, non-specific uptake of a macromolecule by a tumouris enough to deliver an appropriate level of the enzyme in an ADEPT-typeof system and an adequate ratio of tumour-associated cytotoxic drug tonon-tumour-associated drug can be achieved if the enzyme-macromoleculeconjugate is cleared or inhibited when away from the tumour. Thisapproach is applicable to any of the ADEPT systems described above butshould perhaps be called MDEPT (macromolecule directed enzyme prodrugtherapy).

The term “tumour” is to be understood as referring to all forms ofneoplastic cell growth, including tumours of the lung, liver, bloodcells, skin, pancreas, stomach, colon, prostate, uterus, breast, lymphglands and bladder. Solid tumours are especially suitable.

The potential advantages in using non-antibody macromolecules for thispurpose are substantial. Firstly, a non-specific macromolecule may beselected that is non-immunogenic. Secondly, a macromolecule may be muchless costly to produce than humanised antitumour antibody. Thirdly, ithas been shown that some polymers reduce or eliminate the immunogenicityof proteins, including enzymes, attached to them (Abuchowski et al,1977, Wileman el al, 1986, Mikolajczyk et al, 1996). Fourthly, whereasan antibody binds to only a limited range of cancers (antibodies onlyexist for about 60% of all malignancies), the macromolecule uptake bytumours appears to be a characteristic common to all solid cancers sofar examined.

Thus, many tumours such as sarcomas for which no selective antibodieshave yet been reported may be targeted using this principle.

The relatively low differential between tumour and non-tumour tissueswith non-specific macromolecules is exploitable only if the level ofnormal tissue enzyme is inhibited, for example by using a galactosylatedanti-enzyme antibody. To get the required amount of enzyme to tumoursites when the enzyme is conjugated to a non-specific macromolecule mayrequire a greater amount of such a conjugate to be administered thanwould be the case with a specific antibody-enzyme conjugate, but thelower cost of the former should offset its lower efficiency.

Preferably, the macromolecule used in the invention is hydrophilic andis characterised by being soluble in body fluids and in conventionalfluids for parenteral administration. Suitably, the macromolecule isbiodegradable so that systemic accumulation during repeatedadministration is avoided. Clearly, however, it must not be degraded sofast as to fail to accumulate at the tumour site. Preferably, whenconjugated to the selected enzyme, the molecular weight and size of theconjugate should exceed that of the renal threshold for urinaryexcretion (MW 60000), as this helps the blood concentration to besufficient to provide an effective blood:tumour concentration gradient.A molecular weight of up to at least 800000 is generally suitable, forexample up to 160000. The macromolecule is preferably one which is notreadily captured by the reticuloendothelial system. To make itcatalytic, the macromolecule may be conjugated to one or more enzymemolecules by simple chemical methods, using bi-functional agents whichdo not to degrade the attached enzyme. Preferably, the startingmacromolecule confers reduced immunogenicity on an immunogenic enzyme towhich it is conjugated.

Macromolecules that are available as subunits and are not biodegradablemay be linked by biodegradable linking units so that thenon-biodegradable components are filtered through the kidneys andexcreted in the urine.

Whereas some macromolecules are not known to be intermalised by cellsothers, such as N-(2-hydroxypropyl)methylacrylamide, are internalisedthrough more than one mechanism (Duncan et al, 1996).

Preferably, the macromolecule is polyethylene glycol (PEG).Derivatisation of proteins with polyethylene glycol has beendemonstrated numerous times to increase their blood circulationlifetimes as well as decrease their antigenicity and immunogenicity.MDEPT is described in more detail in our co-pending patent applicationPCT/GB97/03284 incorporated herein by reference.

Thus, a preferred embodiment for delivery of the enzyme to tumour sitesis to take advantage of the leakiness of tumour capillaries and the poorlymphatic drainage of tumours. Thus, it has been shown that an enzymeconjugated to form a macromolecule, for instance by conjugation topolyethylene glycols or dextrans is selectively taken up by tumours.

The cDNA encoding human NAD(P)H:quinone reductase 2 (NQO2) is given inJaiswal et al (1990) Biochemistry 29, 1899-1906 and the gene structureof the NQO2 gene is given in Jaiswal (1994) J. Biol. Chem. 269,14502-14508, both of which are incorporated herein by reference and thenucleotide sequence and encoded amino acid sequence is given in FIG. 6.The skilled person can readily obtain and manipulate DNA encoding NQO2based on the teachings contained herein using genetic engineering andrecombinant DNA techniques which are well known, some of which aredescribed in Sambrook et al (1989), “Molecular cloning, a laboratorymanual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

A variant or fragment or fusion or derivative of NQO2 may be used inplace of NQO2 with the given amino acid sequence provided that it hassubstantially the same activity as NQO2 towards a given prodrug. Theenzyme NQO2 catalyses the conversion of, for example, the prodrug CB1954 and prodrug analogues thereof. Thus, preferably the variant orfragment or fusion or derivative of NQO2 has substantially the sameactivity towards CB 1954 as does NQO2 itself. Conveniently, the saidvariant or fragment or fusion or derivative has at least 0.1× thek_(cat)/K_(m) of NQO2, more preferably at least 0.5× and still morepreferably at least 0.9× the k_(cat)K_(m) of NQO2.

Preferably, the variant or fragment or fusion or derivative of NQO2 alsohas substantially the same cofactor specificity. Preferably the variantor fragment or fusion or derivative of NQO2 can use nicotinamideriboside (reduced) (NRH) as a cofactor. Preferably, the variant orfragment or fusion or derivative of NQO2 binds the cofactor at least0.1× as well as NQO2 itself, more preferably at least 0.5× as well andstill more preferably at least 0.9× as well.

By a “variant” we include polypeptides in which one or more amino acidshave been replaced or deleted. Typically, the variant has amino acidconservative replacements in which, for example, the following groups ofamino acid may be interchanged: Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn,Gin; Ser, Thr, Lys, Arg; and Phe, Tyr. Such variants may be made usingthe methods of protein engineering and site-directed mutagenesis. Theterm “variants” also includes polypeptides with insertions anddeletions.

By a “fragment” we mean a portion of NQO2 provided that it retainssubstantially the same activity as NQQ2 towards a given prodrug.

By a “fusion” we mean a fusion of NQO2 or a variant or fragment thereofto any other polypeptide, for example, in some circumstances it may bedesirable to fuse NQO2 or a variant or fragment thereof to anotherpolypeptide which can facilitate purification. This may be, for example,glutathione-S-transferase, or the well known Myc tag sequence or His_(n)where n>4. In each case the additional polypeptide allows the fusion tobe purified by affinity chromatography.

When the two portions of the compound of the first aspect of theinvention are polypeptides they may be linked together by any of theconventional ways of cross-linking polypeptides, such as those generallydescribed in O'Sullivan et al (1979) Anal. Biochem. 100, 100-108. Forexample, an antibody portion may be enriched with thiol groups and theenzyme portion reacted with a bifunctional agent capable of reactingwith those thiol groups, for example the N-hydroxysuccinimide ester ofiodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate(SPDP). Amide and thioether bonds, for example achieved withm-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stablein vivo than disulphide bonds.

It may not be necessary for the whole NQO2 to be present in the compoundof the first aspect of the invention but, of course, the catalyticportion must be present.

Alternatively, the compound may be produced as a fusion compound byrecombinant DNA techniques whereby a length of DNA comprises respectiveregions encoding the two portions of the compound of the inventioneither adjacent to one another or separated by a region encoding alinker peptide which does not destroy the desired properties of thecompound. Conceivably, the two portions of the compound may overlapwholly or partly. The antibody (or other polypeptide portion whichtargets a cell) component of the fusion must be represented by at leastone binding site. Examples of the construction of antibody (or anti-bodyfragment)-enzyme fusions are disclosed by Neuberger et al (1984) Nature312, 604.

The DNA is then expressed in a suitable host to produce a polypeptidecomprising the compound of this aspect of the invention. Thus, the DNAencoding the polypeptide constituting the compound of this aspect of theinvention may be used in accordance with known techniques, appropriatelymodified in view of the teachings contained herein, to construct anexpression vector, which is then used to transform an appropriate hostcell for the expression and production of the polypeptide of theinvention. Such techniques include those disclosed in U.S. Pat. No.4,440,859 issued 3 Apr. 1994 to Rutter et al, U.S. Pat. No. 4,530,901issued 23 Jul. 1985 to Weissman, U.S. Pat. No. 4,582,800 issued 15 Apr.1986 to Crowl, U.S. Pat. No. 4,677,063 issued 30 Jun. 1987 to Mark etal, U.S. Pat. No. 4,678,751 issued 7 Jul. 1987 to Goeddel, U.S. Pat. No.4,704,362 issued 3 Nov. 1987 to Itakura et al, U.S. Pat. No. 4,710,463issued 1 Dec. 1987 to Murray, U.S. Pat. No. 4,757,006 issued 12 Jul.1988 to Toole, Jr. et al, U.S. Pat. No. 4,766,075 issued 23 Aug. 1988 toGoeddel et al and U.S. Pat. No. 4,810,648 issued 7 Mar. 1989 to Stalker,all of which are incorporated herein by reference.

The DNA encoding the polypeptide constituting the compound of thisaspect of the invention may be joined to a wide variety of other DNAsequences for introduction into an appropriate host. The companion DNAwill depend upon the nature of the host, the manner of the introductionof the DNA into the host, and whether episomal maintenance orintegration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognised bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance. Alternatively, the gene for such selectable traitcan be on another vector, which is used to co-transform the desired hostcell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdis-closed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus sublilis), yeasts (for example Saccizaromycescerevisiae), filamentous fungi (for example Aspergillus), plant cells,animal cells and insect cells.

The vectors include a procaryotic replicon, such as the ColE1 ori, forpropagation in a procaryote, even if the vector is to be used forexpression in other, non-procaryotic, cell types. The vectors can alsoinclude an appropriate promoter such as a procaryotic promoter capableof directing the expression (transcription and translation) of the genesin a bacterial host cell, such as E. coli, transformed therewith.

A promoter is an expression control element formed by a DNA sequencethat permits binding of RNA polymerase and transcription to occur.Promoter sequences compatible with exemplary bacterial hosts aretypically provided in plasmid vectors containing convenient restrictionsites for insertion of a DNA segment of the present invention.

Typical procaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329available from Biorad Laboratories, (Richmond, Calif., USA) and pTrc99Aand pKK223-3 available from Pharmacia, Piscataway, N.J., USA.

A typical mammalian cell vector plasmid is pSVL available fromPharmacia, Piscataway, N.J., USA. This vector uses the SV40 latepromoter to drive expression of cloned genes, the highest level ofexpression being found in T antigen-producing cells, such as COS-1cells.

An example of an inducible mammalian expression vector is pMSG, alsoavailable from Pharmacia. This vector uses the glucocorticoid-induciblepromoter of the mouse mammary tumour virus long terminal repeat to driveexpression of the cloned gene.

Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and aregenerally available from Stratagene Cloning Systems, La Jolla, Calif.92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are YeastIntegrating plasmids (YIps) and incorporate the yeast selectable markershis3, trp1, leu2 and ura3. Plasmids pRS413-416 are Yeast Centromereplasmids (YCps).

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive termini. For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted to thevector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors The DNAsegment, generated by endonuclease restriction digestion as describedearlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNApolymerase I, enzymes that remove protruding, 3′-single-stranded terminiwith their 3′-5′-exonucleolytic activities, and fill in recessed 3′-endswith their polymerizing activities.

The combination of these activities therefore generates blunt-ended DNAsegments. The blunt-ended segments are then incubated with a large molarexcess of linker molecules in the presence of an enzyme that is able tocatalyze the ligation of blunt-ended DNA molecules, such asbacteriophage T4 DNA ligase. Thus, the products of the reaction are DNAsegments carrying polymeric linker sequences at their ends. These DNAsegments are then cleaved with the appropriate restriction enzyme andligated to an expression vector that has been cleaved with an enzymethat produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources includingInternational Biotechnologies Inc, New Haven, Conn., USA.

A desirable way to modify the DNA encoding the polypeptide of thisaspect of the invention is to use the polymerase chain reaction asdisclosed by Saiki et al (1988) Science 239, 487-491.

In this method the DNA to be enzymatically amplified is flanked by twospecific oligonucleotide primers which themselves become incorporatedinto the amplified DNA. The said specific primers may containrestriction endonuclease recognition sites which can be used for cloninginto expression vectors using methods known in the art.

Exemplary genera of yeast contemplated to be useful in the practice ofthe present invention are Pichia, Saccharomyces, Kluyveromyces, Candida,Torulopsis, flansenula, Schizosaccharomyces, Citeromyces, Pachysolen,Debaromyces, Metscliunikowia, Rhodosporidium, Leucosporidium,Botryoascus, Sporidiobolus, Endomycopsis, and the like. Preferred generaare those selected from the group consisting of Pichia, Saccharomyces,Kluyveromyces, Yarrowia and Hansenula. Examples of Saccilaromyces areSaccharoinyces cerevisiae, Saccharomyces italicus and Saccliaromycesrouxii. Examples of Kluyveromyces are Kluyveromyces fragilis andKluyveromyces lactis. Examples of Hansenula are Hansenula polyniorpha,Hansenula anomala and Hansenula capsulata. Yarrowia lipolytica is anexample of a suitable Yarrowia species.

Methods for the transformation of S. cerevisiae are taught generally inEP 251 744, EP 258 067 and WO 90/01063, all of which are incorporatedherein by reference.

Suitable promoters for S. cerevisiae include those associated with thePGK1 gene, GAL1 or GAL10 genes, CYC1, PHO5, TRP1, ADH1, ADH2, the genesfor glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, triose phosphate isomerase,phosphoglucose isomerase, glucokinase, α-mating factor pheromone,a-mating factor pheromone, the PRB1 promoter, the GUT2 promoter, andhybrid promoters involving hybrids of parts of 5′ regulatory regionswith parts of 5′ regulatory regions of other promoters or with upstreamactivation sites (eg the promoter of EP-A-258 067).

The transcription termination signal is preferably the 3′ flankingsequence of a eukaryotic gene which contains proper signals fortranscription termination and polyadenylation. Suitable 3′ flankingsequences may, for example, be those of the gene naturally linked to theexpression control sequence used, ie may correspond to the promoter.

Alternatively, they may be different in which case the terminationsignal of the S. cerevisiae AHD1 gene is preferred.

By “polynucleotide encoding said NQO2 or said variant or fragment orfusion or derivative” we include any such polynucleotide. Thepolynucleotide may be RNA or DNA; preferably it is DNA.

It will be appreciated that when the compound of the first aspect of theinvention comprises a polynucleotide encoding human NQO2 or apolynucleotide encoding a variant or fragment or fusion or derivativethereof which has substantially the same activity as NQO2 towards agiven prodrug the target cell-specific portion of the compound is onewhich is adapted to deliver the polynucleotide (genetic construct) tothe target cell.

Preferably, the genetic construct is adapted for delivery to a cell,preferably a human cell. More preferably, the genetic construct isadapted for delivery to a cell in an animal body, more preferably amammalian body; most preferably it is adapted for delivery to a cell ina human body.

Means and methods of introducing a genetic construct into a cell in ananimal body are known in the art. For example, the constructs of theinvention may be introduced into the tumour cells by anv convenientmethod, for example methods involving retroviruses, so that theconstruct is inserted into the genome of the tumour cell. For example,in Kuriyama et al (1991) Cell Struc. and Func. 16, 503-510 purifiedretroviruses are administered. Retroviruses provide a potential means ofselectively infecting cancer cells because they can only integrate intothe genome of dividing cells; most normal cells surrounding cancers arein a quiescent, non-receptive stage of cell growth or, at least, aredividing much less rapidly than the tumour cells. Retroviral DNAconstructs which contain a suitable promoter segment and apolynucleotide encoding NQO2 or a variant or fragment or fusion orderivative as defined may be made using methods well known in the art.To produce active retrovimis from such a construct it is usual to use anecotropic psi2 packaging cell line grown in Dulbecco's modified Eagle'smedium (DMEM) containing 10% foetal calf serum (FCS). Transfection ofthe cell line is conveniently by calcium phosphate co-precipitation, andstable transformants are selected by addition of G418 to a finalconcentration of 1 mg/ml (assuming the retroviral construct contains aneo^(R) gene). Independent colonies are isolated and expanded and theculture supernatant removed, filtered through a 0.45 μm pore-size filterand stored at −70°. For the introduction of the retrovirus into thetumour cells, it is convenient to inject directly retroviral supernatantto which 10 μ/ml Polybrene has been added. For tumours exceeding 10 mmin diameter it is appropriate to inject between 0. 1 ml and 1 ml ofretroviral supernatant; preferably 0.5 ml.

Alternatively, as described in Culver et al (1992) Science 256,1550-1552, cells which produce retroviruses are injected into thetumour. The retrovirus-producing cells so introduced are engineered toactively produce retroviral vector particles so that continuousproductions of the vector occurred within the tumour mass in situ. Thus,proliferating tumour cells can be successfully transduced in vivo ifmixed with retroviral vector-producing cells.

Targeted retroviruses are also available for use in the invention; forexample, sequences conferring specific binding affinities may beengineered into preexisting viral env genes (see Miller & Vile (1995)Faseb J. 9, 190-199 for a review of this and other targeted vectors forgene therapy).

Other methods involve simple delivery of the construct into the cell forexpression therein either for a limited time or, following integrationinto the genome, for a longer time. An example of the latter approachincludes (preferably tumour-cell-targeted) liposomes (Nässander et al(1992) Cancer Res. 52, 646-653).

Inmunoliposomes (antibody-directed liposomes) are especially useful intargeting to cancer cell types which overexpress a cell surface proteinfor which antibodies are available (see Table for examples). For thepreparation of inmmuno-liposomes MPB-PE(N-[4-(p-maleimidophenyl)-butyryl]-phosphatidylethanolamine) issynthesised according to the method of Martin & Papahadjopoulos (1982)J. Biol. Chem. 257, 286-288. MPB-PE is incorporated into the liposomalbilayers to allow a covalent coupling of the antibody, or fragmentthereof, to the liposomal surface. The liposome is conveniently loadedwith the DNA or other genetic construct of the invention for delivery tothe target cells, for example, by forming the said liposomes in asolution of the DNA or other genetic construct, followed by sequentialextrusion through polycarbonate membrane filters with 0.6 μm and 0.2 μmpore size under nitrogen pressures up to 0.8 MPa. After extrusion,entrapped DNA construct is separated from free DNA construct byultracentrifugation at 80000×g for 45 min. Freshly preparedMPB-PE-liposomes in deoxygenated buffer are mixed with freshly preparedantibody (or fragment thereof) and the coupling reactions are carriedout in a nitrogen atmosphere at 4° C. under constant end over endrotation overnight. The immunoliposomes are separated from unconjugatedantibodies by ultracentrifugation at 80000×g for 45 min. Immunoliposomesmay be injected intraperitoneally or directly into the tumour.

Other methods of delivery include adenoviruses carrying external DNA viaan antibody-polylysine bridge (see Curiel Prog. Med. Virol. 40, 1-18)and transferrin-polycation conjugates as carriers (Wagner et al (1990)Proc. Natl. Acad. Sci. USA 87, 3410-3414). In the first of these methodsa polycation-antibody complex is formed with the DNA construct or othergenetic construct of the invention, wherein the antibody is specific foreither wild-type adenovirus or a variant adenovirus in which a newepitope has been introduced which binds the antibody. The polycationmoiety binds the DNA via electrostatic interactions with the phosphatebackbone. It is preferred if the polycation is polylysine.

The DNA may also be delivered by adenovirus wherein it is present withinthe adenovirus particle, for example, as described below.

In the second of these methods, a high-efficiency nucleic acid deliverysystem that uses receptor-mediated endocytosis to carry DNAmacromolecules into cells is employed. This is accomplished byconjugating the iron-transport protein transferrin to polycations thatbind nucleic acids. Human transferrin, or the chicken homologueconalbumin, or combinations thereof is covalently linked to the smallDNA-binding protein protamine or to polylysines of various sizes througha disulfide linkage. These modified transferrin molecules maintain theirability to bind their cognate receptor and to mediate efficient irontransport into the cell. The transferrin-polycation molecules formelectrophoretically stable complexes with DNA constructs or othergenetic constructs of the invention independent of nucleic acid size(from short oligonucleotides to DNA of 21 kilobase pairs). Whencomplexes of transferrin-polycation and the DNA constructs or othergenetic constructs of the invention are supplied to the tumour cells, ahigh level of expression from the construct in the cells is expected.

High-efficiency receptor-mediated delivery of the DNA constructs orother genetic constructs of the invention using the endosome-disruptionactivity of defective or chemically inactivated adenovirus particlesproduced by the methods of Cotten et al (1992) Proc. Natl. Acad. Sci.USA 89, 6094-6098 may also be used. This approach appears to rely on thefact that adenoviruses are adapted to allow release of their DNA from anendosome without passage through the lysosome, and in the presence of,for example transferrin linked to the DNA construct or other geneticconstruct of the invention, the construct is taken up by the cell by thesame route as the adenovirus particle.

This approach has the advantages that there is no need to use complexretroviral constructs; there is no permanent modification of the genomeas occurs with retroviral infection; and the targeted expression systemis coupled with a targeted delivery system, thus reducing toxicity toother cell types.

It may be desirable to locally perfuse a tumour with the suitabledelivery vehicle comprising the genetic construct for a period of time;additionally or alternatively the delivery vehicle or genetic constructcan be injected directly into accessible tumours.

It will be appreciated that “naked DNA” and DNA complexed with cationicand neutral lipids may also be useful in introducing the DNA of theinvention into cells of the patient to be treated. Non-viral approachesto gene therapy are described in Ledley (1995) Human Gene Therapy 6,1129-1144.

Thus, it will be appreciated that a further aspect of the inventionprovides a composition comprising genetic construct as defined in theinvention and means for introducing said genetic construct into a cell,preferably the cell of an animal body.

Alternative targeted delivery systems are also known such as themodified adenovirus system described in WO 94/10323 wherein, typically,the DNA is carried within the adenovirus, or adenovirus-like, particle.Michael et al (1995) Gene Therapy 2, 660-668 describes modification ofadenovirus to add a cell-selective moiety into a fibre protein. Mutantadenoviruses which replicate selectively in p53-deficient human tumourcells, such as those described in Bischoff et al (1996) Science 274,373-376 are also useful for delivering the genetic construct of theinvention to a cell. Thus, it will be appreciated that a further aspectof the invention provides a virus or virus-like particle comprising agenetic construct of the invention. Other suitable viruses or virus-likeparticles include HSV, AAV, vaccinia and parvovirus.

It will be appreciated that in the first aspect of the invention thepolynucleotide need not be one which has a target cell-specific promoterto drive the expression of NQO2 or said variant or fragment or fusion orderivative thereof since the compound comprises a target cell-specificportion as described above for targeting the polynucleotide to thetarget cell. However, it may be advantageous if the polynucleotidecomprises a target cell-specific promoter operably linked to apolynucleotide encoding human NAD(P)H:quinone reductase 2 (NQO2) or avariant or fragment or fusion or derivative thereof which hassubstantially the same activity as NQO2 towards a given prodrug.

It will be further appreciated that target cell-specific expression ofNQO2 or the said variants, fragments, fusions or derivatives may be toachieved using a polynucleotide or genetic construct comprising a targetcell-specific promoter whether or not the polynucleotide or geneticconstruct is comprised in a compound of the first aspect of theinvention.

Thus, a second aspect of the invention provides a recombinantpolynucleotide comprising a target cell-specific promoter operablylinked to a polynucleotide encoding human NAD(P)H:quinone reductase 2(NQO2) or a variant or fragment or fusion or derivative thereof whichhas substantially the same activity as NQO2 towards a given prodrug.

Preferably the target cell-specific promoter is a tumour cell-specificpromoter.

Useful genetic elements which are target cell-specific promoters aregiven below but new ones are being discovered all of the time which willbe useful in this embodiment of the invention.

The tyrosinase and TRP-1 genes both encode proteins which play key rolesin the synthesis of the pigment melanin, a specific product ofmelanocytic cells. The 5′ ends of the tyrosinase and tyrositase-relatedprotein (TRP-1) genes confer tissue specificity of expression on genescloned downstream of these promoter elements.

The 5′ sequences of these genes are described in Bradl, M. et al (1991)Proc. Natl. Acad. Sci. USA 88, 164-168 and Jackson, I. J. et al (1991)Nucleic Acids Res. 19, 3799-3804.

Prostate-specific antigen (PSA) is one of the major protein constituentsof the human prostate secretion. It has become a useful marker for thedetection and monitoring of prostate cancer. The gene encoding PSA andits promoter region which directs the prostate-specific expression ofPSA have been described (Lundwall (1989) Biochem. Biophys. Res. Comm.161, 1151-1159; Riegman et al (1989) Biochein. Biophys. Res. Comm. 159,95-102; Brawer (1991) Acia Oncol. 30, 161-168).

Carcinoembryonic antigen (CEA) is a widely used tumour marker,especially in the surveillance of colonic cancer patients. Although CEAis also present in some normal tissues, it is apparently expressed athigher levels in tumorous tissues than in corresponding normal tissues.The complete gene encoding CEA has been cloned and its promoter regionanalysed. A CEA gene promoter construct, containing approximately 400nucleotides upstream from the translational start, showed nine timeshigher activity in the adenocarcinoma cell line SW303, compared with dieHeLa cell line. This indicates that cis-acting sequences which conveycell type specific expression are contained within this region (Schreweet al (1990) Mol. Cell. Biol. 10, 2738-2748).

The mucin gene, MUC1, contains 5′ flanking sequences which are able todirect expression selectively in breast and pancreatic cell lines, butnot in non-epithelial cell lines as taught in WO 91/09867.

The alpha-fetoprotein (AFP) enhancer may be useful to drive pancreatictumour-selective expression (Su et al (1996) Hum. Gene Ther. 7,463-470).

The genetic constructs of the invention can be prepared using methodswell known in the art.

A third aspect of the invention provides a therapeutic system (or, as itmay be termed, a kit of parts) comprising a compound of the first aspectof the invention or a polynucleotide of the second aspect of theinvention and a prodrug which is converted to a substantially cytotoxicdrug by the action of NQO2.

Preferably the prodrug is CB 1954 or an analogue thereof; mostpreferably the prodrug is CB 1954.

Analogues of CB1954 are suitably defined as molecules which retain theessential structural features of CB1954 ie a benzene ring containing anaziridine ring, two NO₂ groups and another substituent R but whichdiffer in either the relative orientation of the substituents and/or inthe nature of R. A number of analogues have been disclosed in Khan A. H.and Ross W. C. J. (1969) Chem. Biol. Interact. 1, 27-47 and in Khan A.H. and Ross W. C. J. (1971) Chem. Biol. Interact. 4, 11-22, both ofwhich are incorporated herein by reference and in particular the detailsof the analogues of CB1954 are included in this description.

Preferably the therapeutic system further comprises NRH or an analoguethereof which is able to pass reducing equivalents to NQO2. Suitableanalogues include the reduced form of 1-methylnicotinamide and otherswhich are also described in Friedlos et al (1992b) and Knox et al(1995), both of which are incorporated herein by reference.

A fourth aspect of the invention provides a method of treating a patientwith a target cell to be destroyed the method comprising (a)administering to the patient a compound of the first aspect of theinvention or a recombinant polynucleotide of the second aspect of theinvention; (b) allowing the NQO2 or a variant or fragment or fusion orderivative thereof to localize at, or be expressed in, the target cell;and (c) administering a prodrug which is converted to a substantiallycytotoxic drug by the action of NQO2. It is particularly preferred ifNRH or another suitable cofactor of NQO2 is administered to the patient.Another suitable cofactor of NQO2 includes analogues of NRH which areable to pass reducing equivalents to NQO2 and includes molecules whichare able to bind NQO2 and pass reducing equivalents to NQO2substantially as NRH. The administration of the cofactor may be beforeor after or at the same time as administration of the prodrug.

It is particularly preferred if NRH or an analogue thereof isadministered before the prodrug.

Thus, the method is useful in destroying a target cell in a host (egpatient).

Preferably, the patient to be treated has a tumour.

The prodrug may be any suitable prodrug as described above.

Preferably the prodrug is CB 1954 or an analogue thereof.

Preferably, when the compound is one comprising a target cell-specificportion and human NAD(P)H:quinone reductase 2 (NQO2) or a variant orfragment or fusion or derivative thereof which has substantially thesame activity as NQO2 towards a given prodrug, the compound isadministered and, once there is an optimum balance between the targetcell to normal cell ratio of the compound and the absolute level ofcompound associated with the target, the prodrug which is converted to asubstantially cytotoxic drug by the action of NQO2 is administered. Theinterval between the administration of the compound and the prodrug willdepend on the target cell localisation characteristics of the particularcompound, but typically it will be between 6 and 48 hours.

Suitably, prodrug administration commences as soon as the plasmaactivity of enzyme and, by inference, the activity in normal tissues, isinsufficient to catalyse enough prodrug to cause toxicity.

Thus, in a preferred embodiment, NQO2 is conjugated to a monoclonalantibody directed at a tumour-associated antigen so as to localise attumour sites and CB1954 given when the enzyme has cleared from blood andnormal tissues. As discussed above, it is particularly preferred if NRHor another suitable cofactor of NQO2 is administered to the patient.Preferably, NRH or an analogue thereof is administered before theprodrug.

Preferably, when the compound is one comprising a target cell-specificportion and a polynucleotide encoding human NAD(P)H:quinone reductase 2(NQO2) or a variant or fragment or fusion or derivative thereof whichhas substantially the same activity as NQO2 towards a given prodrug, thecompound is administered and, once the NQO2 or a said variant orderivative or fusion or fragment thereof is expressed in the target cellto a useful extent, the prodrug is administered. As discussed above itis particularly preferred if NRH or another suitable cofactor of NQO2 isadministered to the patient. Preferably, NRH or an analogue thereof isadministered before the prodrug.

In this embodiment, the interval between the administration of thecompound and the prodrug will depend on the target cell localisationcharacteristics of the particular compound but also on the expressioncharacteristics of the polynucleotide in the particular target cell.

Preferably, then a recombinant polynucleotide of the second aspect ofthe invention is administered in the method of treatment of theinvention, the recombinant polynucleotide is expressed in the targetcells to produce NQO2 or a said variant or derivative or fragment orfusion thereof and when the expression is at a useful level, the prodrugis administered. As discussed above, it is particularly preferred if NRHor another suitable cofactor of NQO2 is administered to the patient.Preferably, NRH or an analogue thereof is administered before theprodrug.

Thus, the cytotoxic drug is released in relatively high concentration atthe target or tumour site but not at non-target or non-tumour sites.

It will be appreciated that it is not necessary for the compound of theinvention to locate to, or the polynucleotide of the invention to beexpressed in, all target cells but that the compound should locate to,or the polynucleotide be expressed in, sufficient target cells to have adesirable effect upon administration of the prodrug.

At least with the ADEPT-type embodiment of the invention it may beadvantageous to make use of a modification of the system which allowsfor improved target cell selectivity (especially tumour cellselectivity) by clearing antibody-enzyme conjugates from the blood.

The principle of this improvement is described in detail in WO 89/10140,incorporated herein by reference. Thus, clearance of residual enzymeactivity from blood and normal tissues can be accelerated therebymaximising the tumour to normal tissue ratio of enzyme. Acceleratedclearance has been achieved, for example, by means of a monoclonalantibody directed at any part of the antibody-enzyme conjugate but isespecially effective when the anti-enzyme antibody inactivates theenzyme. To avoid the anti-enzyme antibody inactivating enzyme at tumoursites it can be galactosylated which results in rapid removal of theanti-enzyme-enzyme-antibody complex from the blood by galactosereceptors in hepatocytes. This has been described in WO 89/10140 and inSharma et al, 1990.

At least with the ADEPT- and MDEPT-type embodiments of the presentinvention it may be advantageous to make use of a modification of thesystem which allows for improved target cell selectivity (especiallytumour cell selectivity) by using inhibitors of NQO2. For example,flavones are inhibitors of NQO2. Quercetin(3,5,7,3′,4′-pentahydroxyflavone) is the most potent inhibitor tested sofar. It is a competitive inhibitor with respect to NRH (K_(i)=27 nm),and so may be particularly useful. The principle of this improvement isdescribed in detail in our co-pending patent application PCT/GB96/03000,incorporated herein by reference. Thus, an alternative to the use of asecond monoclonal antibody for clearance of enzyme from blood and normaltissues is to employ a small molecule which complements the active siteof the enzyme but is not a substrate and is sterically bound in thesite. Such a molecule has to be administered at a dose level toinactivate enzyme in blood and normal tissues but at a dose levelinsufficient to inactivate the higher concentration of enzyme in tumourtissues.

At least in the ADEPT-type embodiment of the method of the invention itmay be advantageous if the compound is taken up by the target cell suchthat the enzyme is present within the target cell. The principles ofthis improvement are described in our co-pending patent applicationPCT/GB96/03254, incorporated herein by reference.

The methods of treatment of the fourth aspect of the invention allow forthe NQO2 or said derivative or fragment or variant or fusion to bepresent either within the target cell or outside lie target cell. Forexample, in the embodiments wherein a polypeptide version of NQO2 areadministered to the patient the polypeptide may locate either within thetarget cell (for example, by using the ADEPT system with internalisingantibodies) or it may locate outside the target cell (for example, byusing the ADEPT system with antibodies which remain substantiallyoutside the target cell).

Similarly, in the embodiments wherein a polynucleotide encoding NQO2 areadministered to the patient the polynucleotide may express NQO2 which isretained within the target cell or it may express NQO2 outside of, butassociated with, the target cell. External expression of the enzyme maybe achieved by linking it to a signal sequence which directs the enzymeto the surface of a mammalian cell. This will normally be a mammaliansignal sequence or a derivative thereof which retains the ability todirect the enzyme to the cell surface. Suitable signal sequences includethose found in transmembrane receptor tyrosine kinases such as c-erbB2signal sequence, the sequence of which is published in Coussens et al(1985) Science 230, 1132-1139, incorporated herein by reference.

In those embodiments of the method where NQO2 is located outside thetarget cell, but nevertheless associated with die target cell, it willbe appreciated that co-substrate need not be permeable to the cellmembrane and this is a preferred property of the co-substrate in thisembodiment since there will be no reduction of the prodrug byendogenous, intracellular NQO2. In this embodiment it is preferred ifthe prodrug is substantially unable to permeate the cell membranealthough it may do so. However, it is believed that the cytotoxic drugshould be able to penetrate the cell because it is generally believedthat its cytotoxic effect is due to its reactivity within the cell.

It is preferred if the system of the third aspect of the inventionfurther comprises a cosubstrate for NQO2 which is substantiallypermeable to the target cell membrane.

It is also preferred if the method of the fourth aspect of the inventionfurther comprises administering to the patient an effective amount of aco-substrate for NQO2 which can substantially permeate to the targetcell membrane.

It is preferred if the co-substrate is NRH or an analogue thereofespecially one which can substantially permeate a cell membrane.Suitable analogues include the reduced form of 1-methylnicotinamide andothers which are also described in Friedlos et al (1992b) and Knox et al(1995), both of which are incorporated herein by reference.

Thus, it will be seen that the compound of the first aspect of theinvention and the recombinant polynucleotide of the second aspect of theinvention are useful in medicine and that they are therefore packagedand presented for use in medicine.

The invention also provides the use of a compound of the first aspect ofthe invention, or a polynucleotide of the second aspect of theinvention, in the manufacture of a medicament for treating a, targetcell to be destroyed. Preferably the patient has been, is being or willbe administered a prodrug which is converted to a substantiallycytotoxic drug by action of NQO2.

The invention also provides the use of a prodrug which is converted to asubstantially cytotoxic drug by the action of NQO2 in the manufacture ofa medicament for treating a patient with a target cell to be destroyedwherein the patient has been, is being or will be administered acompound according to the first aspect of the invention, or apolynucleotide according to the second aspect of the invention.

The invention also provides the use of NRH or an analogue thereof whichcan pass reducing equivalents to NQO2 in the manufacture of a medicamentfor treating a patient with a target cell to be destroyed wherein thepatient has been, is being or will be a compound according to the firstaspect of the invention, or a polynucleotide according to the secondaspect of the invention and a prodrug which is converted to asubstantially cytotoxic drug by the action of NQO2.

A fifth aspect of the invention provides a method of treating a humanpatient with a target cell to be destroyed wherein the target cellexpresses NQO2 the method comprising administering to the patient aprodrug which is converted to a substantially cytotoxic drug by theaction of NQO2 and nicotinamide riboside (reduced) (NRH) or an analoguethereof which can pass reducing equivalents to NQO2.

Preferably the target cell expresses NQO2 naturally (for example byvirtue of the disease state) but it may be a target cell which has beeninduced to produce NQO2 or which expresses NQO2 by virtue of inductionor manipulation of the cell.

The NRH analogues are those as described above in relation to theprevious aspects of the invention.

The prodrugs are those as described above in relation to the previousaspects of the invention. It is particularly preferred if the prodrug isCB1954 or an analogue thereof.

It is particularly preferred if NRH or an analogue thereof which issubstantially able to permeate the target cell membrane is used in themethod of the fifth aspect of the invention.

Preferably the target cell is a tumour cell. It is particularlypreferred to use the method of treatment for tumours which show anelevated level of NQO2 compared to non-tumour tissue.

Co-administration of CB 1954 with the co-factor NRH provides a basis foractivation of CB 1954 at intracellular sites where NQO2 is expressed.Present indications are that the enzyme is highly expressed incolorectal cancers. It may also be expressed in some normal tissues and,if so, activation in normal tissues may be dose limiting.

Thus, it is particularly preferred to treat colorectal cancers with themethod of the invention.

According to the method of the invention a prodrug and a co-substrate isadministered to a tumour-bearing mammalian host. The prodrug, that ismuch less cytotoxic to tumour cells than the active drug, is convertedto its active form by the enzyme human NAD(P)H:quinone oxidoreductase 2(NQO2) only in the presence of the co-substrate. Prodrugs that areuseful in the method of this invention include, but are not limited to,CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). Co-substrates that areuseful in the method of this invention include, but are not limited to,NRH (nicotinamide mononucleoside-reduced (dihydronicotinamide riboside))(FIG. 2). It is appreciated that both the prodrug and co-substrateshould be substantially capable of permeating the cell membrane. NADHand NMNH are substantially impermeable to cell membranes. However, itwill be appreciated that “by administering NRH or an analogue thereof”in relation to this and previous aspects of the invention we includeadministering a compound which is converted within the body of thepatient to NRH or an analogue thereof. It will be appreciated that afurther embodiment includes the possibility of administering to thepatient a precursor of NRH or an analogue thereof and means forconverting the precursor to NRH or an analogue thereof.

According to a preferred embodiment of this invention endogenous NQO2 isused to activate CB 1954 in the presence of NRH (FIG. 3). Using in vitroenzyme assays it is demonstrated that CB 1954 is reduced to its4-hydroxylamine derivative(5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide). This reduction ismuch greater than that by either human or rat DT-diaphorase and is notreadily catalysed by either of the biogenic co-substrates, NADH orNADPH.

The method of the fifth aspect of the invention is particularly suitedfor the treatment of a patient with target cells to be destroyed whereinthe target cells express NQO2. Thus, in a particularly preferredembodiment it is determined whether the target cells express NQO2 priorto administration of the prodrug or NRH or an analogue. Thisdetermination can be achieved, for example, by measuring NQO2 levels ina sample comprising the target cell. This may be achieved enzymaticallyor by using probes selective for the NQO2 polypeptide or mRNA.Conveniently, this can be achieved using the techniques commonlyreferred to as western and northern blotting, respectively. In the caseof the polypeptide the probe may be a mono- or polyclonal antibodyraised against the NQO2 protein or a fragment thereof. Such antibodiescould also be used to identify NQO2 in tissue sections by usingimmunocytochemistry and related techniques. Probes against mRNA will beoligonucleotides or DNA fragments complementary to partial sequences ofthe NQO2 MRNA sequence. Although these methods are preferred, othermethods may be used to detect and quantify NQO2 polypeptide or MnRNAlevels in a target cell or tissue.

The invention therefore also includes a therapeutic system comprising aprodrug which is converted to a substantially cytotoxic drug by theaction of NQO2 and nicotinamide riboside (reduced) (NRH) or an analoguethereof which can pass reducing equivalents to NQO2. It is preferred ifthe system further comprises means for determining whether the targetcell expresses NQO2.

The invention also includes nicotinamide riboside (reduced) (NRH) or ananalogue thereof which can pass reducing equivalents to NQQ2 for use inmedicine, use of nicotinamide riboside (reduced) (NRH) or an analoguethereof which can pass reducing equivalents to NQO2 in the manufactureof a medicament for treating a human patient with a target cell to bedestroyed, and use of a prodnig which is converted to a substantiallycytotoxic drug by the action of NQO2 in the manufacture of a medicamentfor treating a human patient with a target cell to be destroyed whereinthe patient has been, is being or will be administered NRH or ananalogue thereof which can pass reducing equivalents to NQO2.

By “NRH or an analogue thereof which is able to pass reducingequivalents to NQO2” we include, as is mentioned above, the reduced formof 1-methylnicotinamide and others which are also described in Friedloset al (1992b) and Knox et at (1995). The k_(cat) for NQO2/NRH is 360min⁻¹ using CB1954 as an electron acceptor. A co-substrate (ie ananalogue of NRH which is able to pass reducing equivalents to NQO2) is acompound that can act as a co-substrate for NQO2 so that the enzyme canreduce CB1954 to its 4-hydroxylamine derivative with a k_(cat)>50 min⁻¹.For the avoidance of doubt, an “analogue of NRH which is able to passreducing equivalents to NQO2” need not necessarily be a structuralanalogue of NRH but is a functional analogue of NRH in the sense that itcan pass reducing equivalents to NQO2. For the avoidance of doubt NADHand NADPH are not co-substrates for NQO2.

Certain cosubstrates (analogues of NRH) have the structure:

where R¹ is alkyl, aryl, substituted alkyl, substituted aryl,CONR^(a)R^(b) (where R^(a) and R^(b) are independently H, alkyl, orsubstituted alkyl), and R² and R³ are independently H, alkyl, orsubstituted alkyl. R⁴ is any of H, alkyl, substituted alkyl, halogen,CN, COOH, CONH₂ or OH. Preferably, R⁴ is H.

Whether or not compounds of this structure act as cosubstrates of NQO2can readily be determined by methods as disclosed herein.

Preferably, R² and R³ are H. Preferably R¹ is alkyl or substitutedalkyl. Thus, it is preferred if the co-substrate has the generalstructure

where R is alkyl or substituted alkyl.

It is preferred that the alkyl group is C₁ to C₆ alkyl, and it isfurther preferred that the alkyl group is a linear alkyl group.

By “substituted alkyl” we include substitution by OH, halogen, CN, COOHand CONH₂.

We have synthesised the following compounds:

-   1: R═—CH₂CH₂CH₂SO₃-   2: R═—CH₂CONH₂-   3: R═—CH₂CH₂CH₃-   4: R═—CH(CH₃)₂-   5: R═—CH₂CH₂CH₂OH-   6: R═—CH₂CH₂OH-   7: R═—CH₂CH₂COOH-   8: R═—CH₂C₆H₅-   9: R═—CH₃-   10: R═—CH₂CH₃-   11: R═—CH₂CH₂C₆H₅

For MDEPT compound 1 is preferable. It is charged at physiological pHand is excluded from cells.

Compound 8 and compound 11 are not cosubstrates for NQO2 by ourdefinition.

It is expected that charged or polar compounds (eg compounds 1, 7) willnot readily enter cells. Lipophilic derivatives (eg compounds 3, 4) areexpected to readily enter cells.

The invention will now be described in more detail by reference to thefollowing Examples and Figures wherein

FIG. 1 shows the bioactivation of CB 1954. The initial step is thereduction of CB 1954 by the enzyme DT diaphorase to form5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. This hydroxylaminederivative can react with thioesters to produce DNA reactive species. Itis postulated that this it the N-acetoxy derivative. The major productof this reaction is however 4-amino-5-(aziridin-1-yl)-2-nitrobenzamidethat does not react readily with DNA. Formation of4-amino-5-(aziridin-1-yl)-2-nitrobenzamide is in competition with theproduction of DNA binding products.

FIG. 2 shows the structure of NRH.

FIG. 3 is a schematic representation of the bioactivation of CB 1954 byNQO2.

FIG. 4 shows the reduction of CB 1954 by NQO2 in the presence of variouscosubstrates.

FIG. 5 shows the formation of5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide (4-NHOH) from thereduction of CB 1954 by NQO2.

FIG. 6 shows the nucleotide sequence of a cDNA encoding human NQO2 andits deduced amino acid sequence (SEQ ID NO:2.

FIG. 7 shows the structures of potential co-substrates for NQO2.

FIG. 8 a shows the ability of NQO2 to use NRH and various analogues asco-substrates for the reduction of CB 1954. In the absence of enzymethere was no CB1954 reduction (not shown). The initial concentration ofco-substrate was 500 μm and the enzyme concentration was 1 μg/ml.

FIG. 8 b shows the ability of NQO2 to use NRH and various analogues asco-substrates for the reduction of CB 1954. In the absence of enzymethere was no CB1954 reduction (not shown). The initial concentration ofco-substrate was 500 μm and the enzyme concentration was 0.5 μg/ml.

FIG. 8 c shows the ability of NQO2 to use NRH and various analogues asco-substrates for the reduction of CB 1954. In the absence of enzymethere was no CB1954 reduction (not shown). The initial concentration ofco-substrate was 500 μm and the enzyme concentration was 0.5 μg/ml.

FIG. 9 shows the uptake of various co-substrates into wild-type V79cells. Compound 1 is charged at physiological pH and is excluded fromthe cells. Thus this co-substrate is particularly suitable for MDEPTapplications.

FIGS. 10A and 10B show the plasmids pIRES-P and H6, respectively.

FIG. 11 shows the effect of NRH on the cytotoxicity of CB 1964 in NQO2expressing V79 cells. The addition of NRH increased the cytotoxicity ofCB 1954 by at least 100-fold (V79TM13) and was greater than 100-fold inthe V79TM5 and 13 cell lines. This effect was not seen innon-transfected V79 cells (<3-fold) and can thus be ascribed to theexpression of NQO2 in the transfected cells.

FIG. 12 shows the effect of NRH, compound 1 and compound 2 on thecytotoxicity of CB 1954 in human T98G gliobastoma cells. The cells weretreated as for V79 cells but were treated for 144 hr in presence of CB1954. The addition of NRH and compound 2 increased the cytotoxicity ofCB 1954 by at least 100-fold whilst the impermeable co-substratecompound 1 did not potentiate.

FIG. 13 shows the ability of rat DT diaphorase to utilise compounds 1and 2 as co-substrates for the reduction of CB1954.

FIG. 14 shows the ability of E. coli nitroreductase to utilise compounds1 and 2 as co-substrates for the reduction of CB1954.

FIGS. 15 and 16 show the effect of compound 1 on the body weight ofnormal mice.

FIGS. 17 and 18 show the effect of compound 2 on the body weight ofnormal mice. Mice [6 groups of 3] were injected intravenously (tailvein) with either 1 or 2 at the doses shown and the weight of the micewas monitored over an 8 day period. Control mice received vehicle[phosphate buffered saline] only.

EXAMPLE 1 The Effect of Various Co-substrates on Human NQO2 Activity

Experimental Details

Recombinant human NQO2 was prepared in E. coli. A NcoI and a HindIIIrestriction site were added to the 5′- and 3′-ends of the full-lengthNQO2 cDNA, respectively, using a PCR method with primers and nucleotidesequences derived from the 5′ and 3′-ends of the cDNA. The PCR productwas resolved over a 1% agarose gel and then extracted using the QIAquickGel Extraction Kit (Qiagen Inc). The gel-purified PCR product was clonedinto PCRII vector from the TA cloning kit (invitrogen Co) and thecorrect sequence of the PCR product was checked by dideoxy sequencing.The resulting construct was religated into the E. coli expressionvector, pKK233-2 (Pharmnacia) through the engineered NcoI and HindIIIrestriction sites. The expression plasmid was designated pKK-hNQO2. ThepKK-hNQO2 E. coli cells were cultured, sonicated and centrifuged aspreviously described for the purification of recombinant DT diaphorase[Chen et al, 1992]. The supernatant from a 90 min-centrifugation at105,00 g was applied to a 50 ml Affi-gel Blue (Bio-Rad) and the columnwas washed according to the published method. The purified NQO2preparation was analysed by SDS-PAGE electrophoresis. The activity ofNQO2 in the presence of CB 1954, and various cofactors was determined byHPLC. To determine the kinetic parameters NQO2 (1 μg/ml) was incubatedwith NRH (500 μM) and CB 1954 at different concentrations (0.1 to 2 mM)in sodium phosphate buffer (10 mM, pH7) at 37° C. At various times,aliquots (10 μl) were injected onto a Partisphere SCX (250×4.5 mm) HPLCcolumn (Whatman Ltd) and eluted isocratically (1.5 ml/min) with 50 mMaqueous sodium phosphate containing 1% methanol. The eluate wascontinuously monitored for absorption at 320 nm. This separation systemcould resolve all the expected reduction products of CB1954 [Boland etal, 1991, Knox et al, 1992]. The reduction of CB 1954 was monitored byquantifying the increase in the area of the peak corresponding to thereduction product 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide.All the assays were initiated by addition of the enzyme and performed induplicate. The kinetic parameters were calculated by plotting theinitial rate of reduction at each concentration of CB 1954 against thatconcentration and fitting the data to the Michales-Menton equation usinga computer programme (FigP). Values were confirmed by transforming thedata and fitting it to various linear forms of the equation byregression analysis.

The effect of various co-substrates on CB 1954 was determined as abovebut NADH. NADPH or NMNH was substituted for the NRH and CB 1954 was usedat a fixed concentration of 100 μM. The enzyme concentration was 5μg/ml. The reduction of CB 1954 was monitored by measuring both thedecrease in its corresponding peak area on the HPLC trace and theincrease in the area of the peak corresponding to the reduction product5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. The relative ratesof reduction were determined at 10% reduction of CB1954 from a graphplotting CB 1954 reduction against time. The time axis was normalised tothe equivalent of 10 μg/ml of NQO2.

Table 1. Kinetic parameters for NQO2, E. coli nitroreductase, human andrat DT-diaphorase with respect to CB 1954. NRH was used as aco-substrate for NQO2 whilst the values for the other enzymes weredetermined using NADH.

ENZYME Km (μM) k_(cat) (s⁻¹) NQO2 263 ± 13  6.01 Nitroreductase¹ 862 ±145 6.0 Rat DT-diaphorase²  826 0.0683 Human DT-diaphorase² 1403 0.0107Data from: ¹Anlezark et al, 1992 ²Boland et al, 1991

Table 2. The relative rate of CB 1954 reduction by NQO2 using differentco-substrates. All co-substrates were used at an initial concentrationof 500 μM and CB 1954 was at an initial concentration of 100 μM.

RELATIVE RATE CO-SUBSTRATE OF REDUCTION NADH 1.0 NADPH 1.24 NMNH 5.6 NRH70.0

EXAMPLE 2 Administration of a Monoclonal Antibody-NQO2 Conjugate

In this example the prodrug is administered 6-48 hours followingadministration of a monoclonal antibody-NQO2 conjugate. The exactinterval depends upon the localisation characteristics of the conjugatebut prodrug administration ideally commences as soon as the plasmaactivity of enzyme is insufficient to catalyse enough prodrug to causetoxicity. The dose of conjugate is in the range 100-300 mg m⁻² perpatient. Administration of the co-substrate NRH commences approximately1 hour prior to the administration of prodrug and continues throughoutthe period of prodrug administration. The dose of prodrug depends uponits nature but an effective dose may be in the range 10-2000 mg m⁻². Thedose of NRH may be 2-3 times the dose of prodrug. In this system it maybe advantageous to accelerate clearance of residual enzyme activity fromplasma and normal tissues. This may be achieved by administration of agalactosylated anti-enzyme antibody following conjugate administrationbut prior to administration of NRH

EXAMPLE 3 Administration of a Recombinant NQO2 Polytiucleotide

In this example a recombinant polynucleotide is administered.Administration may be by any route appropriate to the condition to betreated, suitable routes including oral, nasal and parenteral. Thedosage is determined by the individual clinicians for individualpatients and this is determined by the exact nature of the prodrug andthe cytotoxic agent to be released from the prodrug. Approximate dosesare given in Example 2 above. When the expression of NQO2 is at a usefullevel administration of NRH followed by prodrug can commence as detailedin Example 2.

EXAMPLE 4 Administration of NRH and Prodrug

In this example a patient is administered NRH and after 1 hourconcurrent administration of prodrug is started. As previously, thedoses of prodrug and NRH will depend upon the nature of the prodrug andthe cytotoxic agent released from the prodrug.

EXAMPLE 5 Potential Co-substrates for NQO2

The compounds synthesised are shown in FIG. 7.

Details of the syntheses are now given.

Compound 1: 1-(3-sulfonatopropyl)-dihydronicotinamide

To a solution of 1-(3-sulfonatopropyl)-3-carboxamidopyridinium (20 mg)in water (5 mL) was added 50 mg of anhydrous sodium carbonate, 50 mg ofsodium bicarbonate and 50 mg of sodium hydrosulphite and the stopperedsolution was allowed to stand at 37° for 30 min. The reduced compoundwas purified from the reaction mixture by preparative HPLC. 5 ml ofreaction mixture was injected onto a Dynamax Macro C18 (21.4×250 mm)reverse-phase column (Rainin) and eluted by a gradient of acetonitrilein water (0-100% over 30 min) at 10.0 ml/min. The eluate was continuallymonitored at 340 nm and by fluorescence (ex 340, em 450) and a fractioncorresponding to a peak of fluorescence collected. The eluate wascollected and freeze-dried to provide compound 1. NMR (D₂O, 270 MHz, 20°C.) d 1.95-2.08 (m, J˜7.3 Hz, 2H, NCH₂CH₂CH₂SO₃ ⁻), 2.94 (t, J=7.7 Hz,2H, NCH₂CH₂CH2SO₃ ⁻), 3.04 (br t, J=1.8 Hz, 1H, 4-CH₂), 3.34 (t, J=6.8Hz, 2H, NCH₂CH₂CH₂SO₃ ⁻), 4.88-4.98 (m, 1H, H-5), 5.95 (dd, J =8.1 Hz,J=1.5 Hz, 1H, H-6), 7.04 (s, 1H, H-2).

The starting material 1-(3-sulfonatopropyl)-3-carboxamidopyridinium wasprepared as follows:

1,3-Propanesultone (12.21 g, 0.10 mol) was added in one portion to astirred solution of nicotinamide (12.21 g, 0.10 mol) inN,N-dimethylformamide (DMF,20 cm³). The clear solution was heated to100° C. for 1 h, during which time (>5 min) a heavy colourless solidseparated. The reaction mixture was cooled to room temperature,filtered, and the solid was washed serially with cold DMF (2×25 cm³)then dry diethyl ether (2×30 cm³). Recrystallisation from aqueous DMFgave 1-(3-sulfonatopropyl)-3-carboxamidopyridinium as colourless prisms:mp 300-302° C. dec; NMR (D₂O, 270 MHz, 20° C.) d 2.52 (quintet, J=7.3Hz, 2H, N⁺CH₂CH₂CH₂SO₃ ⁻), 3.04 (t, J=7.3 Hz, 2H, N⁺CH₂CH₂CH₂SO₃ ⁻),4.89 (t, J=7.3 Hz, 2H, N⁺CH₂CH₂CH₂SO₃ ⁻), 7.95 (br s, slow exchange,CONH₂), 8.24 (br t, J ⁻7.2 Hz, 1H, H-5), 8.94 (d, J=8.1 Hz, 1H, H4),9.10 (d, J=6.2 Hz, 1H, H-6), 9.40 (s, 1H, H-2). Found: C, 43.38; H,4.95; N: 10.94%. C₉H,₁₂N₂O₄S·0.25H₂O (anhydrous M=244.27) requires C,43.45, H, 5.06; N, 11.26%.

Compound 2

Compound 2, 1-(carboxamidomethyl)-dihydronicotinamide, was prepared from1-(carboxamidomethyl)-3-carboxamidopyridinium iodide following thereduction procedure described for compound 1.

NMR (D₂O, 270 MHz, 20° C.) d 3.00 (br t, J=1.8 Hz, 2H, 4-CH₂) 3.90 (s,2H, CH₂CONH₂), 4.82-4.90 (m, 1H, H-5), 5.76 (dm, J=8.1 Hz, H-6), 6.89(s, 1H, H-2).

The starting material 1-(carboxamidomethyl)-3-carboxamidopyridiniumiodide was prepared by heating a mixture of nicotinamide (2.0 g, 16.4mmol) and 2-iodoacetamide (3.1 g, 16.8 mmol) in DMF (5 ml) at 55-60° C.for 3 h. After cooling to room temperature ethyl acetate (50 ml) wasadded and the mixture was stirred for 30 min. The product was removed byfiltration, dried at the pump and recrystallised from aqueous ethanol togive 1-(carboxamidomethyl)-3-carboxamidopyridinium iodide as colourlesscrystals (3.3 g, 66%): mp 210-211° C. Found: C, 31.37, H, 3.34 ; N,13.77%. C₈H₁₀N₃0₂I (anhydrous M =307.09) requires C, 31.29; H, 3.28; N,13.68%.

Compound 3

Compound 3, 1-propyl-dihydronicotinamide, was prepared from1-propyl-3-carboxanidopyridinium bromide following the reductionprocedure described for compound 1. NMR (CDCl₃, 270 MHz, 20° C.) d 0.90(t, J=7.3 Hz, 3H, NCH₂CH₂CH₃), 1.56 (sextet, J=7.3 Hz, 2H, NCH₂CH₂CH₃),3.05 (t, J=7.3 Hz, 2H, NCH₂CH₂CH₃), 3.16 (dd, J =3.6 Hz, J=1.8 Hz, 2H,4-CH₂), 4.72 (dt, J=8.1 Hz, J=3.6 Hz, 1H, H-5), 5.35 (br s, 2H, slow D₂Oexchange, CONH₂), 5.72 (dq, J=8.1 Hz, 1.8 Hz, 1H, H-6), 7.04 (d, J=1.8Hz, H-2).

The starting material 1-propyl-3-carboxamidopyridinium bromide wasprepared as follows. A solution of nicotinamide (12.21 g, 0.10 mol) and1-bromopropane (12.30 g, 0.10 mol) in DMF (20 cm³) was stirred andheated to 70° C. for 1 h. A heavy precipitate appeared within 15 min.After cooling to room temperature overnight, the mixture was filteredand the solid washed with cold DMF (10 cm³) then dry diethyl ether (2×20cm³). Recrystallisation from DMF gave 1-propyl-3-carboxamidopyridiniumbromide (19.42 g, 79%) as colourless prisms: mp 171.5-172.5° C.; NMR(d₆-DMSO, 270 MHz, 20° C.) d 0.91 (t, J=7.3 Hz, 3H, N⁺CH₂CH₂CH₃), 2.01(sextet, J=7.3 Hz, 2H, N⁺CH₂CH₂CH₃), 4.70 (t, J=7.3 Hz, 2H, N^(+CH)₂CH₂CH₃), 8.20 (br s, slow D₂O exchange, 1H, CONH_(a)H_(b)), 8.32 (dd,J=8.1 Hz, J=6.2 Hz, 1H, H-5), 8.68 (br s, slow D₂O exchange, 1H,CONH_(a)H_(b)), 9.02 (d, J=8.1 Hz, 1H, H-4), 9.35 (d, J=6.2 Hz, 1H,H-6), 9.65 (s, 1H, H-2). Found: C, 44.21; H, 5.36; N: 11.32%. C₉H₁₃N₂OBr (anhydrous M =245.12) requires C, 44.10, H, 5.35; N, 1 1.43%.

Compound 3 could also be prepared by reduction of1-propyl-3-carboxamidopyridinium iodide. This starting material wasprepared by heating a mixture of nicotinamide (2.0 g, 16.4 mmol) and1-iodopropane (3.2 ml, 32.8 mmol) in DMF (5 ml) at 90-95° C. for 4 h.Ethyl acetate (50 ml) was added to the cooled solution and the mixturewas stirred at room temperature for 30 min. The solid was filtered,dried at the pump and recrystallised from methanol to give1-propyl-3-carboxamidopyridinium iodide as pale yellow crystals (2.3 g,48%): mp 183-184° C. (lit. 180-182° C. [S. Liao, J. T. Dulaney & H. G.Williams-Ashman, J Biol Chem., 237, 2981-2987 (1962)]; NMR (d₆-DMSO, 270MHz, 20° C.) δ0.90 (t, 3H), 1.98 (m, 2H), 4.64 (t, 2H), 8.17 (s, 1H),8.30 (t, 1H), 8.54 (s, 1H), 8.94 (d, 1H), 9.23 (s, 1H), 9.49 (s, 1H).Found: C, 37.05; H, 4.47; N, 9.49%. C₉H₁₃N₂OI (anhydrous M=292.12)requires C, 37.01; H, 4.49; N, 9.59%.

Compound 4

Compound 4, 1-(2-propyl)dihydronicotinamide, was prepared from1-(2-propyl)-3-carboxamidopyridinium bromide following the reductionprocedure described for compound 1.

The starting material 1-(2-propyl)-3-carboxamidopyridinium bromide wasprepared as follows. A solution of nicotinamide (12.21 g, 0.10 mol) and2-bromopropane (12.30 g, 0.10 mol) in DMF (20 cm³) was stirred andheated to 70° C. for 10 h, during which time a colourless precipitateappeared. After cooling to room temperature overnight, the mixture wasfiltered and the solid washed with cold DMF (10 cm³) then dry diethylether (2×20 cm³). Recrystallisation from DMF gave1-(2-propyl)-3-carboxamidopyridinium bromide (16.81 g, 69%) ascolourless prisms: mp 215.5-217.0° C.; NMR (d₆-DMSO, 270 MHz, 20° C.) d1.68 (d, J=7.0 Hz, 6H, N⁺CH(CH₃)₂), 5.17 (septet, J=7.0 Hz, 2H,N⁺CH(CH₃)₂), 8.20 (br s, slow D₂O exchange, 1H, CONH₃H_(b)), 8.31 (dd,J=8.1 Hz, J=6.2 Hz, 1H, H-5), 8.71 (br s, slow D₂O exchange, 1H,CONH_(a)H_(b)), 8.98 (d, J=8.1 Hz, 1H, H-4), 9.43 (d, J =6.2 Hz, 1H,H-6), 9.62 (s, 1H, H-2). Found: C, 44.19; H. 5.34; N: 11.30%. C₉H₁₃N₂OBr(anhydrous M=245.12) requires C, 44.10, H, 5.35; N, 11.43%.

Compound 4 could also be prepared by reduction of1-(2-propyl)-3-carboxamidopyridinium iodide. This was prepared byheating a mixture of nicotinamide (2.0 g, 16.4 mmol) and 2-iodopropane(3.0 ml, 30.0 mmol) in DMF (5.0 ml) at 90-95° C. for 4 h. Ethyl acetate(50 ml) was added to the cooled solution and the mixture was stirred atroom temperature for 30 min. The mixture was filtered and the solid wasdried at the pump and recrystallised from aqueous ethanol to give1-(2-propyl)-3-carboxamidopyridinium iodide as yellow crystals (1.1 g,23%): mp 188-189° C. Found: C, 37.16; H, 4.55; N, 9.53%. C₉ H₁₃N₂OI(anhydrous M=292.12) requires C, 37.01; H, 4.49; N, 9.59%.

Compound 5

Compound 5, 1-(3-hydroxypropyl)-dihydronicotinamide,was prepared from1-(3-hydroxypropyl)-3-carboxamidopyridinium bromide following thereduction procedure described for compound 1.

NMR (D₂O, 270 MHz, 20° C.) d 1.89 (br quintet, J⁻6.6 Hz, 2H,NCH₂CH₂CH₂OH), 3.17 (br t, J=1.8 Hz, 2H, 4-CH₂), 3.38 (t, J=6.9 Hz, 2H,NCH₂CH₂CH₂OH), 3.74 (t, J=6.2 Hz, 2H, NCH₂CH₂CH₂OH), 4.95-5.05 (m, 1H,H-5), 6.01 (dm, J=8.1 Hz, 1H, H-6), 7.13 (s, 1H, H-2).

The starting material 1-(3-hydroxypropyl)-3carboxamidopyridinium bromidewas prepared as follows. A solution of nicotinamide (12.21 g, 0.10 mol)and 3-bromo-1-propanol (13.90 g, 0.10 mol) in DMF (20 cm³) was stirredand heated to 90° C. for 1 h. After cooling to room temperatureovernight, the mixture was filtered and the solid washed with cold DMF(10 cm³) then dry diethyl ether (2×25 cm³). Recrystallisation from DMFgave 1-(3-hydroxypropyl)-3-carboxamidopyridinium bromide (19.29 g, 74%)as colourless prisms: mp 119.0-120.0° C.; NMR d₆-DMSO, 270 MHz, 20° C.)d 2.14 (br quintet, J⁻6.2 Hz, 2H, N⁺CH₂CH₂CH₂OH), 3.48 (t, J=5.7 Hz, 2H,N⁺CH₂CH₂CH₂OH), 4.77 (t, J=7.0 Hz, 2H, N⁺CH₂CH₂CH₂OH), 8.18 (br s, slowD₂O exchange, 1H, CONH_(a)H_(b)), 8.28 (dd, J=8.1 Hz, J=5.9 Hz, 1H,H-5), 8.63 (br s, slow D₂O exchange, 1H, CONH_(a)H_(b)), 8.97 (d, J=8.1Hz, 1H, H4), 9.26 (d, J=5.9 Hz, 1H, H-6), 9.56 (s, 1H, H-2). Found: C,40.07; H, 5.17; N: 10.16%. C₉H₁₃N₂O₂Br-0.5H₂O (anhydrous M=261.12)requires C, 40.02, H, 5.22; N, 10.37%.

Compound 6

Compound 6, 1-(2-hydroxyethyl)-dihydronicotinamide, was prepared from1-(2-hydroxyethyl)-3-carboxamidopyridinium iodide following thereduction procedure described for compound 1.

The starting material 1-(2-hydroxyethyl)-3-carboxamidopyridinium iodidewas prepared by heating a mixture of nicotinamide (2.0 g, 16.4 mmol) and2-iodoethanol (2.6 ml, 33.3 mmol) in DMF (5 ml) at 90-95° C. for 4 h.Ethyl acetate (50 ml) was added to the cooled solution and the mixturewas stirred at room temperature for 30 min. The mixture was filtered andthe solid was dried at the pump and recrystallised from methanol to give1-(2-hydroxyethyl)-3-carboxamidopyridinium iodide as colourless crystals(3.8 g, 79%): mp 128-129° C. NMR (d₆-DMSO, 270 MHz, 20 ° C.) d 3.90 (brt, J=7.3 Hz, 2H, N⁺CH²CH₂0H), 5.21 (t, J =7.3 Hz, 2H, N⁺CH₂CH₂OH), 8.17(br s, slow D₂O exchange, 1H, CONH_(a)H_(b)), 8.32 (dd, J=8.1 Hz, J=6.2Hz, 1H, H-5), 8.56 (br s, slow D₂O exchange, 1H, CONH_(a)H_(b)), 8.98(d, J=8.1 Hz, 1H, H-4), 9.16 (d, J=6.2 Hz, 1H, H-6), 9.43 (s, 1H, H-2).Found: C, 33.06; H, 3.85; N, 9.57%. C₈H₁₁N₂O₂I (anhydrous M=294.09)requires C, 32.67, H, 3.77; N, 9.53%.

Compound 7

Compound 7, 1-(2-carboxyethyl)-dihydronicotinamide, was prepared from1-(2-carboxyethyl)-3-carboxamidopyridinium iodide according to thereduction procedure described for compound 1.

NMR (D₂O, 270 MHz, 20° C.) 2.46 (t, J=6.9 Hz, 2H, NCH₂CH₂CO₂H), 2.96 (t,J=6.9 Hz, 2H, NCH₂CH₂CO₂H), 4.85-4.95 (m, 1H, H-5), 7.33-7.31 (m,),8.19-8.24 (m), 8.29-9.05 (m), 9.36 (s).

The starting material 1-(2-carboxyethyl)-3-carboxamidopyridinium iodidewas prepared by heating a mixture of nicotinamide (2.0 g, 16.4 mmol) and3-iodopropionic acid (3.3 g, 16.5 mmol) in DMF (5 ml) at 90-95° C. for 4h. Ethyl acetate (50 ml) was added to the cooled solution and themixture was stirred at room temperature for 30 min. The mixture wasfiltered and the solid was dried at the pump and recrystallised fromaqueous ethanol to give 1-(2-carboxyethyl)-3-carboxamidopyridiniumiodide as colourless crystals (2.4 g, 46%): mp 185-186° C. Found: C,33.80; H, 3.45; N, 8.57%. C₉H₁₁N₂O₃I (anhydrous M=322.10) requires C,33.56; H, 3.44; N, 8.70%.

Compound 8

Compound 8 1-benzyl-dihydronicotinamide was prepared from1-benzyl-3-carboxamidopyridinium iodide according to the reductionprocedure described for compound 1.

1-benzyl-3-carboxamidopyridinium bromide was prepared by heating amixture of nicotinamide (2.0 g, 16.4 mmol) and benzyl bromide (3.9 ml,32.8 mmol) in DMF (5 ml) at 55-60° C. After ⁻5 minutes a heavyprecipitate formed and a further portion of DMF (5 ml) was added. After30 min the mixture was cooled to room temperature and ethyl acetate (50ml) was added. The mixture was stirred at room temperature for 30 minthen the solid was filtered, dried at the pump and recrystallised fromaqueous ethanol to give 1-benzyl-3-carboxamidopyridinium bromide ascolourless prisms (4.2 g, 85%): mp 212-213° C. NMR (d₆-DMSO, 270 MHz, 20° C.) d 5.99 (s, 2H, N⁺CH₂Ph), 7.35-7.55 (m, 3H, H-3′/4′/5′), 7.55-7.70(m, 2H, H-2′/6′) 8.22 (br s, slow D₂O exchange, 1H, CONH_(a)H_(b)), 8.32(dd, J=8.1 Hz, J=6.2 Hz, 1H, H-5), 8.68 (br s, slow D₂O exchange, 1H,CONH_(a)H_(b)), 9.03 (d, J=8.1 Hz, 1H, H-4), 9.39 (d, J=6.2 Hz, 1H,H-6), 9.74 (s, 1H, H-2). Found: C, 53.35; H, 4.48; N: 9.40%. C₁₃H₁₃N₂OBr(anhydrous M=293.16) requires C, 53.26; H, 4.47; N, 9.56%.

Compound 9

Compound 9: 1-methyl-dihydronicotinamide was prepared from1-methyl-3-carboxamidopyridinium iodide according to the reductionprocedure described for compound 1

The starting material 1-methyl-3-carboxamidopyridinium iodide, wasobtained from Sigma-Aldrich Chemical Company, Poole Dorset, UK. NMR(d₆-DMSO, 270 MHz, 20° C.) d 4.41 (s, 3H, N⁺CH₃), 8.16 (br s, slow D₂Oexchange, 1H, CONH_(a)H_(b)), 8.26 (dd, J=8.4 Hz. J=6.2 Hz, 1H, H-5),8.52 (br s, slow D₂O exchange, 1H, CONH_(a)H_(b)), 8.91 (d, J=8.4 Hz,1H, H-4), 9.12 (d, J=6.2 Hz, 1H, H-6), 9.41 (s, 1H, H-2).

Compound 10

Compound 10, 1-ethyl-dihydronicotinamide, was prepared from1-ethyl-3-carboxamidopyridinium iodide according to the reductionprocedure described for compound 1.

The starting material 1-ethyl-3carboxamidopyridinium iodide was preparedby heating a mixture of nicotinamide (2.0 g, 16.4 mmol) and 1-iodoethane(2.6 ml, 32.5 mmol) in DMF (5 ml) at 55-60° C. for 3 h. After cooling,ethyl acetate (50 ml) was added and the mixture was stirred at roomtemperature for 30 min. The mixture was filtered and the pale yellowsolid was dried at the pump and recrystallised from a mixture of DMF andethyl acetate to give 1-ethyl-3-carboxamidopyridinium iodide as paleyellow prisms (3.7 g, 82%): mp 202-203° C. NMR (d₆-DMSO, 270 MHz, 20 °C.) d 1.59 (t, J=7.3 Hz, 3H, N⁺CH₂CH₃), 4.72 (q, J=7.3 Hz, 2H,N⁺CH₂CH₃), 8.16 (br s, slow D₂O exchange, 1H, CONH_(a)H_(b)), 8.30 (dd,J=8.1 Hz, J=6.2 Hz, 1H, H-5), 8.53 (br s, slow D₂O exchange, 1H,CONH_(a)H_(b)), 8.93 (d, J=8.1 Hz, 1H, H-4), 9.27 (d, J=6.2 Hz, 1H,H-6), 9.50 (s, 1H, H-2). Found: C, 34.11; H, 3.91; N: 9.80%. C₈H₁₁N₂OI(anhydrous M=278.09) requires C, 34.55; H, 3.99; N, 10.07%.

Compound 11

Compound 11, 1-phenylethyl-dihydronicotinamide, was prepared from1-phenylethyl-3-carboxamidopyridinium iodide according to the reductionprocedure described for compound 1.

The starting material 1-phenylethyl-3-carboxamidopyridinium iodide wasprepared by heating a mixture of nicotinamide (2.0 g, 16.4 mmol) and(2-iodoethyl) benzene (4.7 ml, 32.5 mmol) in DMF (5 ml) at 55-60° C. for4 h. Ethyl acetate (50 ml) was added to the cooled solution and themixture was stirred at room temperature for 30 min. The solid wasfiltered, dried at the pump and recrystallised from aqueous ethanol togive 1-phenylethyl-3-carboxamidopyridinium iodide as yellow prisms (3.8g, 65%): mp 188-189° C. NMR (d₆-DMSO, 270 MHz, 20° C.) d 3.32 (t., J=7.3Hz, 2H, N⁺CH₂CH₂Ph), 4.93 (t, J=7.3 Hz, 2H, N⁺CH₂CH₂Ph), 7.15-7.40 (m,5H, H-2′/3′/4′/5′/6′), 7.55-7.70 (m, 2H, H-2′/6′) 8.17 (br s, slow D₂Oexchange, 1H, CONH_(a)H_(b)), 8.26 (dd, J=8.1 Hz, J=6.2 Hz, 1H, H-5),8.52 (br s, slow D₂O exchange, 1H, CONH_(a)H_(b)), 8.93 (d, J=8.1 Hz,1H, H-4), 9.15 (d, J=6.2 Hz, 1H, H-6), 9.47 (s, 1H, H-2).

We have examined compounds 1-11 as potential co-substrates for NQO2 andwe have determined full kinetic properties for compounds 1 and 2.Experimental details and results are given below.

Compounds 3, 8, 9 and 10 have been reported in the literature (S Liao, JT Dulaney & H G Williams-Ashrman, J. Biol. Chem., 237, 2981-2987, 1962;S Liao & H G Williams-Ashman, Biochem. and Biophys. Res. Commn., 4,208-213, 1961; S Liao & H G Williams-Astuiian, Biochem. Pharmacol., 6,53-54, 1961.)

FIG. 8 a shows that compound 8 and nicotinic acid mononucleotide(reduced) are not co-substrates for NQO2 and that compound 11 is a poorco-substrate. FIG. 4 shows that NADH and NADPH are poor co-substratesfor NQO2.

The k_(cat) of NQO2 with NRH is 360 min⁻¹ using CB 1954 as an electronacceptor. The K_(m) for this reaction is about 30 μM. The new cofactorsare certainly very good at reducing CB 1954 in the presence of NQO2.

Determination of Co-substrate Activity with NQO2

The activity of various potential co-substrates was determined by HPLCanalysis in the presence of CB1954 and NQO2. To determine the kineticparameters NQO2 (1 μg/ml) was incubated with NRH (5000 μM) and CB1954 atdifferent concentrations (0.1 to 2 mM) in sodium phosphate buffer (10mM, pH7) at 37° C. At various times, aliquots (10 μl) were injected ontoa Partisphere SCX (250×4.5 mm) HPLC column (Whatman Ltd) and elutedisocratically (1.5 ml/min) with 50 mM aqueous sodium phosphatecontaining 1% methanol. The eluate was continuously monitored forabsorption at 320 nm. This separation system could resolve all theexpected reduction products of CB1954 [Boland et al, 1991; Knox et al,1992]. The reduction of CB1954 was monitored by quantifying the increasein the area of the peak corresponding to the reduction product5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. All the assays wereinitiated by addition of the enzyme and performed in duplicate. Thekinetic parameters were calculated by plotting the initial rate ofreduction at each concentration of CB1954 against that concentration andfitting the data to the Michaelis-Menton equation using a computerprogramme (Fig P). Values were confirmed by transforming the data andfitting it to various linear forms of the equation by regressionanalysis.

The effect of various co-substrates on CB1954 reduction was determinedas above but the co-substrate was substituted for the NRH and C1954 wasused at a fixed concentration of 100 μM. The enzyme concentration was 1or 5 μg/ml. The reduction of CB1954 was monitored by measuring both thedecrease in its corresponding peak area on the HPLC trace and theincrease in the area of the peak corresponding to the reduction product5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. The relative ratesof reduction were determined at 10% reduction of CB1954 from a graphplotting CB1954 reduction against time. The time axis was normalised tothe equivalent of 10 μg/ml of NQO2. The kinetic parameters of NQO2 forthe various co-substrates was determined as for CB1954 except that theinitial concentration of CB1954 was constant at 100 μm whilst that ofthe co-substrate was varied from 0 to 2 mM. The enzyme concentration was0.5 μg/ml.

FIGS. 8 a-c show the ability of all of the compounds to act ascosubstrates.

The kinetic data determined for compounds 1 and 2 are given in Table 3.

TABLE 3 The kinetic parameters of NQO2 for various co-substrates.Co-substrate K_(m) (μM) k_(cat) (min⁻¹) NRH 28 ± 2   360 2 198 ± 19  750 1 1080 ± 135  1530

EXAMPLE 6 Internalisation of NQO2 Co-substrates into Cells

Compound 1 bears a negative charge so we do not expect it to enter cellsand this is what is observed. Compound 2 seems to be poorly internalised(about 10% of NRH). The poor internalisation may be due to intracellularmetabolism.

The uptake of various NQO2 co-substrates into V79 cells was determinedby fluorimetry. V79 cells were seeded into T25 tissue culture flasks andallowed to grow to confluence (⁻2×10⁷ cells). The growth medium wasremoved and replaced with 10 mls of fresh medium containing 1 mM ofco-substrate and incubated at 37° C. At various the medium was removedand the cell monolayer washed ×5 with 50 mL of ice cold PBS. The cellswere removed by trypsinisation and pelleted by centrifugation. The cellpellet was resuspended in alkaline lysis buffer (KOH 0.5M; 36% w/v CsClin 50% aqueous ethanol) disrupted by agitation and the cell debrisremoved by centrifugation at 10,000 g. The concentration of co-substratewas determined by fluorimetry (excitation 360 nm, emission 450 nm, slits5 nm) by diluting 100 μl of supernatant in 2.9 ml of 100 mM sodiumbicarbonate buffer pH10. The fluorimeter was calibrated against theappropriate standard. All measurements were done in triplicate. Theresults are shown in FIG. 9.

FIG. 9 shows the uptake of various co-substrates into wild-type V79cells. Compound 1 is charged at physiological pH and is excluded fromthe cells. Thus this co-substrate is suitable for MDEPT applications.

EXAMPLE 7 Cytotoxicity of CB1954 in Cells Transfected with NQO2 in thePresence or Co-substrates

We have transfected NQO2 into V79 (Chinese hamster lung embryofibroblasts) cells and looked at the cytotoxicity of CB1954 in thepresence of co-substrates.

Construction of NQO2 Vector H6

The NQQ2 sequence was derived from a bacterial expression plasmidpKK-hNQO2 described in Example 1. Stages in construction were:

1) The NQO2 ORF was excised from pKK-hNQO2 as a NcoI/HindIII fragment,the NcbI site incorporating the start codon.

2) This was cloned into F58 cut NcoI/HindIII to produce the vector H1.F58 is a derivative of pBluescript II SK(+) (Stratagene) produced inhouse and incorporates an extra XhoI site, a Kozak sequence for goodeukaryotic expression and an NcoI site (CCTCGAGTCACCATGGATATCnnn . . . )(SEQ ID NO:1) inserted at the Bluescript EcoRV site.

3) A partial 2 base fill was used to link the 3′ HindIII site of theNQO2 ORF to the MCS XbaI site of die puromycin-resistant IRESbicistronic eukaryotic expression vector F250 (pIRES-P, EMBL:Z75185(FIG. 10 a). H1 and F250 were first cut with XhoI and the plasmid DNApurified. H1 was then cut with HindIII and treated with Klenow DNApolymerase in the presence of dA+dG only, F250 was cut with XbaI andtreated with Klenow and dC+dT only. The XhoI-[XbaI/CT] linearised F250and the XhoI-[HindIII/AG] nqo2 insert were then ligated together toproduce the final expression vector H6 (FIG. 10 b).

Transfection of V79 Cells

Transfection quality H6 DNA was prepared using the QIAGEN endotoxin-freemaxiprep kit. Chinese hamster lung embryo fibroblasts (V79) weretransfected with the purified H6 using DOTAP liposomal reagent accordingto the manufacturer's instructions (Boehringer Mannheim). Two days aftertransfection puromycin-resistant clones were selected in DMEM/10% FCScontaining 10 μg/ml puromycin (Sigma) and grown on to establish celllines designated V79TM1, 5, 3, 7, 9, 11 and 13 respectively andmaintained in selective medium.

Cytotoxicity Analysis in Vitro

Cells in exponential phase of growth were trypsinised, seeded in 96 wellplates at a density of 5×10⁴ cells per well (100 μl) and permitted torecover for 24 hours. Then 50 μl of medium was removed and replaced with50 μl of fresh medium containing 200 μM NRH to give a finalconcentration of 100 μM. Serial dilutions of CB 1954 (8 of 3.66×) wereperformed in situ giving final concentrations of 1000-0.46 μM. Cellswere then incubated with drug for 72 hours at 37° C. The plates werefixed and stained with sulforhodamine-B; the absorption at 590 nm readand results were expressed as percentage of control growth. The IC₅₀values were evaluated by interpolation. As a control, non-transfectedV79 cells were treated as above but in this case the medium did notcontain puromycin. Growth curves are shown in FIG. 11.

FIG. 11 shows the effect of NRH on the cytotoxicity of CB 1964 in NQO2expressing V79 cells. The addition of NRH increased the cytotoxicity ofCB 1954 by at least 100-fold (V79TM13) and was greater than 100-fold inthe V79TM5 and 13 cell lines. This effect was not seen innon-transfected V79 cells (<3-fold) and can thus be ascribed to theexpression of NQO2 in the transfected cells.

EXAMPLE 8 Cytotoxicity of CB1954 on T98G Glioblastoma Cells in thePresence of NQO2 Co-substrate

We have examined the cytotoxicity of CB1954 in the presence of eitherNRH, compound 1 or compound 2 on the T98G glioblastoma cell line. CB1954alone was not toxic to these cells. CB1954 cytotoxicity was increased byat least 100-fold when cells were incubated with CB1954 and either NRHor compound 2. In the presence of compound 1, which is not able to enterthe cells, no potentialtion is observed. The implication is that NQO2 ispresent in the cells and its activation of CB 1954 is the cause of thecytotoxicity.

The results are shown in FIG. 12.

FIG. 12 shows the effect of NRH, compound 1 and compound 2 on thecytotoxicity of CB 1954 in human T98G gliobastoma cells. The cells weretreated as for V79 cells but were treated for 144 hr in presence of CB1954. The addition of NRH and compound 2 increased the cytotoxicity ofCB 1954 by at least 100-fold whilst the impermeable co-substratecompound 1 did not potentiate.

EXAMPLE 9 Selectivity of NQ02 Co-substrates and in vivo Toxicity

Selectivity of the New Co-substrates

FIGS. 13 and 14 show the ability of other CB1954-reducing enzymes (E.coli nitroreductase and rat DT diaphorase) to utilise the newco-subtrates. Unlike NQO2, both of these enzymes can use NADH as aco-substrate to reduce CB1954. The data show that rat DT diaphorase isable to use both compound 1 and compound 2 as co-substrates in thereduction. However, E. coli nitroreductase is not able to use either.Thus the new co-substrates show a degree of selectivity and are notgeneral electron donors.

FIGS. 15 and 16 show the effect of compound 1 on the body weight ofnormal mice.

FIGS. 17 and 18 show the effect of compound 2 on the body weight ofnormal mice. Mice (6 groups of 3) were injected intravenously (tailvein) with either compound 1 or 2 at the doses shown and the weight ofthe mice was monitored over an 8 day period. Control mice receivedvehicle (phosphate buffered saline) only.

Compounds 1 and 2 showed no evidence of intrinsic toxicity to mice asjudged by body weight loss.

References Cited

-   Anlezark, G. M., Melton, R. G., Sherwood, R. F., Coles, B.,    Friedlos, F. and Knox, R. J. (1992) “The bioactivation of    5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)-I. Purification and    properties of a nitroreductase enzyme from Escherichia coli—a    potential enzyme for antibody-directed enzyme prodrug therapy    (ADEPT)” BiochemPharmacol 44, 2289-95.-   Boland, M. P., Knox, R. J. and Roberts, J. J. (1991) “The    differences in kinetics of rat and human DT diaphorase result in a    differential sensitivity of derived cell lines to CB 1954    (5-(aziridin-1-yl)-2,4-dinitrobenzamide)” Biochem Pharmacol 41,    867-75.-   Chen, H. H., Ma, J. X., Forrest, G. L., Deng, P. S., Martino, P. A.,    Lee, T. D. and Chen, S. (1992) “Expression of rat liver    NAD(P)H:quinone-acceptor oxidoreductase in Escilerichia coli and    mutagenesis in vitro at Arg-177” Biochem J. 284, 855-60.-   Chen, S., Knox, R. J., Wu, K., Deng, P. S. K., Zhou, D.,    Biancher, M. A. and Amzel, L. M. (1997) “Molecular basis of the    catalytic differences among DT-diaphorase of human, rat and mouse”    Journal of Biological Chemistry 272, 1437-1439.-   Connors, T. A. and Knox, R. J. (1995) “Prodrugs in medicine” Expert    Opinion on Therapeutic Patents 5, 873-885.-   Connors, T. A. and Whisson, M. E. (1966) “Cure of mice bearing    advanced plasma cell tumours with aniline mustard: the relationship    between glucuronidase activity and tumour sensitivity” Nature 210,    866-7.-   Friedlos, F., Biggs, P. J., Abrahamson, J. A. and Knox, R. J.    (1992a) “Potentiation of CB 1954 cytotoxicity by reduced pyridine    nucleotides in human tumour cells by stimulation of DT diaphorase    activity” Biochem Pharmacol 44, 173-943.-   Friedlos, F., Jannan, M., Davies, L. C., Boland, M. P. and    Knox, R. J. (1992b) “Identification of novel reduced pyridinium    derivatives as synthetic co-factors for the enzyme DT diaphorase    (NAD(P)H dehydrogenase (quinone), EC 1.6.99.2)” Biochem Pharmacol    44, 25-31.-   Friedlos, F. and Knox, R. J. (1992) “Metabolism of NAD(P)H by blood    components. Relevance to bioreductively activated prodrugs in a    targeted enzyme therapy system” Biochem Pharmacol 44, 631-5.-   Jaiswal, A. K. (1994) “Human NAD(P)H:quinone oxidoreductase2. Gene    structure, activity, and tissue-specific expression” J Biol Chem    269, 14502-8.-   Jaiswal, A. K., Burnett, P., Adesnik, M. and Wesley, M. O. (1990)    “Nucleotide and deduced amino acid sequence of a human cDNA (NQO-2)    corresponding to a second member of the NAD(P)H: quinone    oxidoreductase gene family. Extensive polymorphism at the NQO-2 gene    locus on chromosome 6” Biochemistry 29, 1899-1906.-   Knox, R. J., Friedlos, F. and Boland, M. P. (1993) “The    bioactivation of CB 1954 and its use as a prodrug in    antibody-directed enzyme prodrug therapy (ADEPT)” Cancer Metastasis    Rev 12, 195-212.-   Knox, R. J., Friedlos, F., Janman, M., Davies, L. C., Goddard, P.,    Anlezark, G. M., Melton, R. G. and Sherwood, R. F. (1995) “Virtual    cofactors for an Escherichia-coli nitroreductase enzyme—relevance to    reductively activated prodrugs in antibody-directed enzyme prodrug    therapy (adept)” Biochemical Pharmacology 49, 1641-1647.-   Knox, R. J., Friedlos, F., Sherwood, R. F., Melton, R. G. and    Anlezark, G. M. (1992) “The bioactivation of    5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)-II. A comparison of    an Escherichia coli nitroreductase and Walker DT diaphorase” Biochem    Pharmacol 44, 2297-301.-   Mauger, A. B., Burke, P. J., Somani, H. H., Friedlos, F. and    Knox, R. J. (1994) “Self-immolative prodrugs: candidates for    antibody-directed enzyme prodrug therapy in conjunction with a    nitroreductase enzyme” J Med Chem 37, 3452-8.-   Quinn, J. (1996) “Studies on CB1954 and its analogues” PHD Thesis:    Universiry of London.-   Sharma, S. K., Bagshawc, K. D., Burke, P. J., Boden, R. W and    Rogers, G. T. (1990) “Inactivation and clearance of an anti-CEA    carboxypeptidase G2 conjugate in blood after localisation in a    xenograft model” Br. J. Cancer 61, 659-662.-   Whisson, M. E. and Connors, T. A. (1965) “Cure of mice bearing    advanced plasma cell tumours with aniline mustard” Nature 206,    689-91.

1. A method of treating a human patient with a target tumor cell to bedestroyed wherein the target tumor cell expresses NQO2 the methodcomprising administering to the patient a prodrug which is converted toa cytotoxic drug by the action of NQO2 and an analogue of nicotinamideriboside (reduced) (NRH) which can pass reducing equivalents to NQO2,wherein the prodrug is CB 1954, and wherein the analogue ofnicotinamide, riboside (reduced) (NRH) is 1-carboxamidomethyl)dihydronicotinamide.
 2. The method of claim 1 wherein the analogue ofNRH is able to permeate the target cell membrane.
 3. The method of claim1, the method further comprising determining, before administering theprodrug and analogue of NRH, whether the target tumor cell to be treatedexpresses NQO2.